Transcription factor-E2F-5

ABSTRACT

Two novel transcription factors belonging to the E2F gene family, are disclosed. These are human and murine E2F-5. They can interact with DP-1 and p130.

This invention relates to a novel transcription factor and to itsproduction and uses.

The molecular events that occur during the cell cycle need to beintegrated with the transcription apparatus so that gene expression canbe synchronised with cell cycle progression.

Recently, a transcription factor called E2F (or DRTF1) has beenidentified and shown to bind to pRb, the protein product of theretinoblastoma susceptibility gene, an anti-oncogene or tumoursuppressor gene (see for example Wagner and Green, Nature 352, 189-190,1991). It is widely believed that the cellular transcription factor E2Ffunctions as a key component in cell cycle control because it associateswith important cell cycle regulating proteins, such as theretinoblastoma gene product (pRb), p107, cyclins and cyclin-dependentkinases, and furthermore its transcriptional activity is modulated bycertain viral oncoproteins, such as adenovirus Ela, SV40 large Tantigen, and the human papilloma virus E7 protein.

It is believed that the transcription factor E2F (or DRTF1) plays animportant role in integrating cell cycle events with the transcriptionapparatus because, during cell cycle progression in mammalian cells, itundergoes a series of periodic interactions with molecules that areknown to be important regulators of cellular proliferation. For example,the retinoblastoma tumour suppressor gene product (pRb), whichnegatively regulates progression from G1 into S phase. and is frequentlymodified in tumour cells, binds to E2F. Similarly, the pRb-relatedprotein p107 occurs predominantly in an S phase complex with E2F. BothpRb and p107 repress the transcriptional activity of E2F, which islikely to be fundamentally important for regulating cellularproliferation because E2F binding sites occur in the control regions ofa variety of genes that are involved with proliferation, such as c-mycand p34^(cdc2). Furthermore, mutant Rb proteins, encoded by allelesisolated from tumour cells, fail to bind to E2F, and hence are unable tointerfere with E2F site-dependent transcriptional activation. Anotherimportant feature of E2F is that certain viral oncoproteins, such asadenovirus Ela, SV40 large T antigen and human papilloma virus E7,modulate its activity by sequestering pRb and p107 from the inactivetranscription factor. This effect requires regions in these viralproteins that are necessary for transformation of tissue culture cellsand hence to overcome growth control. Thus, the ability of theseoncoproteins to regulate E2F may be the means by which they over-ridethe normal mechanisms of cellular growth control and, conversely,transcriptional repression by pRb may be the basis of pRb-mediatednegative growth control.

A potential mechanism for integrating the transcription-regulatingproperties of pRb and p107 with other cell cycle events was suggested bythe identification of cyclin A and the cdc2-related cyclin-dependentkinase p33^(cdk2) in the E2F complex. Cyclin A is necessary forprogression through S phase, a function that could perhaps be mediatedthrough its ability to recruit the cyclin-dependent kinase p33^(cdk2) toE2F. Taken together these data suggest that E2F is a transcriptionfactor whose primary role may be to relay cell cycle events to thetranscription apparatus via molecules such a pRb, p107, cyclins andcdks, thus ensuring that gene expression is synchronised and integratedwith cell cycle progression.

More recently, a transcription factor with the properties of E2F hasbeen cloned and sequenced (Helin et al, Cell 70 (1992), 337-350 andKaelin et al, Cell 70 (1992), 351-364).

SUMMARY OF THE INVENTION

We have now surprisingly found a further two new proteins which appearto be new members of the E2F gene family, which we have called E2F-5.The cDNA sequence of human E2F-5 is presented in FIG. 1A, as is theamino acid sequence of this protein. The corresponding sequences formurine E2F-5 appear in FIG. 9A. These new proteins are referred to asE2F-5 and this nomenclature will be used in this specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Human E2F-5 structure

(A) Nucleotide sequence and deduced amino acid sequence of the humanE2F-5 cDNA. (SEQ ID NOs: 1 and 2, respectively)

(B) Schematic representation of E2F-5 in comparison with E2F-1 andE2F-4. The borders of the conserved domains are indicated by amino acidnumber. SS in E2F-4 indicates the serine-rich motif.

FIG. 2 Expression pattern of E2F-5 in human cell lines.

(A) Northern blot containing total cytoplasmic RNA from the indicatedhuman cell lines was hybridized to a human E2F-5 cDNA probe. RNAs fromthe follow human cell lines was used: CAMA, human breast carcinoma;A549, lung carcinoma; MDA, MDA-MD157 breast carcinoma; OVCAR, ovariumcarcinoma; HS 578T, breast carcinoma; LUCY, ovarium carcinoma; HT29,colon carcinoma: CEM, T-cell leukemia; K562, erythroleukemia. (B) Thesame filter hybridized with a rar α-tubulin probe.

FIG. 3. E2F-5 has a corboxyl-terminal transactivation domain.

U-2 OS cells were transfected with a CAT reporter plasmid harboringupstream Gal4 sites (5 μg) in the presence or absence of Gal-4-E2Fexpression vectors (1 μg) and 0.2 μg pRSV-Luciferase as an internalcontrol. CAT activiry was normalize to the luciferase activity for eachsample. CAT activity was assayed two days post transfection . The foldactivation of Gal4-E2F over CAT reporter gene alone is represented. Dataare representative for at least three independent experiments done induplicate.

FIG. 4. E2F-5 and DP-1 cooperate in transactivation.

U2-OS osteosarcoma cells were transfected with increasing amounts ofpJ3-E2F-5 expression vector (1, 2, or 5 μg) together with 100 ngpCMV-DP-1, as indicated. In each transfection 2 μg reporter construct(E2F₄-CAT) and 0.2 μg pRSV-Luciferase was added. CAT activity wasnormalized to the luciferase activity for each sample. Data arerepresentative for at least three independent experiments done induplicate.

FIG. 5. Inhibition of E2F-5 transactivation by pocket proteins.

U2-OS cells were transfected with 5 μg pJ3E2F-5 and 100 ng pCMV-DP-1 incombination with pCMV-Rb (50 and 100 ng), pCMV-RbΔ22 (100 ng), pCMV-p107(50 and 100 ng), pCMVp107DE (100 ng) or pCMV-HA-p130 (50, 100 and 500ng). Together with the expression plasmids, the cells were transfectedwith 2 μg E2F₄-CAT and 0.2 μg pRSV-Luciferase. CAT activity wasnormalized to the luciferase internal control. Fold activation wascalculated relative to the basal level of E2F₄-CAT which was set tounity (1.0). Data are representative for at least three independentexperiments done in duplicate.

FIG. 6. E2F containing complexes in transiently transfected U2-OS cells.

U2-OS osteosarcoma cells were transiently transfected with E2F-5 andDP-1 expression vectors in the presence or absence of pRb- p107 or p130expression vectors as indicated. After two days, whole cell extractswere prepared and incubated with a [¹²P]-labeled oligonucleotidecontaining a consensus E2F DNA binding site and subjected to gelelectrophoresis. The position of free probe, E2F-5/DP-1 DNA complex andE2F-5/pocket protein complex is indicated.

FIG. 7. E2F-5 preferentially interacts with p130 in vivo.

Human CAMA breast carcinoma cells were labeled with [³²P]-orthophosphateand non-ionic detergent lysates were subjected to sequentialimmunoprecipitation. In a first immunoprecipitation lysates wereincubated with pRb, p107 or p130 antibody as indicated. Panel A showsthe first immunoprecipitations with pRb- p107 and p130-specificantibodies from CAMA cells. The pRb-, p107 and p130-associated proteinswere released from the immunoprecipitated pocket proteins by boiling inSDS-containing buffer and re-immunoprecipitated with e2F-5-specificantiserum (panel B). As a control, proteins released from pRb- and p107immunoprecipitates were re-immunoprecipitated with anti E2F-1 (KH20) orE2F-4 (RK13) antibody (panel C). Immunoprecipitated proteins wereseparated on 7.5% SDS-polyacrylamide gels, the gels were dried andproteins were detected by autoradiography.

FIG. 8

Strategy for isolating murine E2F-5 (SEQ ID NO:3)

The ‘bait’, LEXA DP-1, was used to screen a 14.5 d.p.c. mouse embryoactivation-domain tagged cDNA library.

FIG. 9

Sequence and comparison of murine E2F-5 with other members of the E2Ffamily.

(a) Nucleotide sequence together with predicted amino acid residuesequence of E2-F5. (SEQ ID NOs: 3 and 4, respectively)

(b) Diagrammatic representation and comparison of E2F-5 (middle) withE2F-1 (top) and E2F-4 (bottom). Percentage identities at the level ofprotein sequence are indicated between E2F-5 and E2F-1, and E2F-5 andE2F-4. Domains shared between E2F family members are indicated (Lees etal., 1993).

(c) Comparison of amino acid residue sequence in the conserved domainswithin the E2F; family members.

(SEQ ID NOs: 5-10) The highlighted residues are common to all familymembers, whereas boxed residues are common to E2F-4 and E2F-5. Boldresidues in the leucine zip region indicate hydrophobics in a heptadrepeat. Residues in the DEF box region which are shared between E2Ffamily member and DP-1 are indicated by the lines. (SEQ ID NOs:11-25)

DNA Binding/Dimerization E2F-1 (SEQ ID NO:5)

DNA Binding/Dimerization E2F-2 (SEQ ID NO:6)

DNA Binding/Dimerization E2F-3 (SEQ ID NO:7)

DNA Binding/Dimerization E2F-4 (SEQ ID NO:8)

DNA Binding/Dimerization E2F-5 (SEQ ID NO:9)

DNA Binding/Dimerization DP-1 (SEQ ID NO:10)

Leucine Zip E2F-1 (SEQ ID NO:11)

Leucine Zip E2F-2 (SEQ ID NO:12)

Leucine Zip E2F-3 (SEQ ID NO:13)

Leucine Zip E2F-4 (SEQ ID NO:14)

Leucine Zip E2F-5 (SEQ ID NO:15)

Marked Box E2F-1 (SEQ ID NO:16)

Marked Box E2F-2 (SEQ ID NO:17)

Marked Box E2F-3 (SEQ ID NO:18)

Marked Box E2F-4 (SEQ ID NO:19)

Marked Box E2F-5 (SEQ ID NO:20)

Pocket Protein Binding E2F-1 (SEQ ID NO:21)

Pocket Protein Binding E2F-2 (SEQ ID NO:22)

Pocket Protein Binding E2F-3 (SEQ ID NO:23)

Pocket Protein Binding E2F-4 (SEQ ID NO:24)

Pocket Protein Binding E2F-5 (SEQ ID NO:25)

FIG. 10

Activation of E2F site-dependent transcription by E2F-5 and DP-1 inyeast.

(a) Summary of reporter and effector constructs.

(b) The indicated E2F-5 and DP-1 expression vectors were transformedinto yeast either alone (lanes 2 and 3) or together (lane 4) and theactivity of p4xWT CYC1, which carries four wild-type E2F sites,assessed. In parallel experiments, the activity of p4xMT CYC1 was notaffected in any of the conditions. The data presented were derived fromtriplicate readings.

FIG. 11

Transcriptional activation by E2F-5 in yeast.

(a) Summary of reporter and effector constructs

(b) The transcription activity of either pLEX.E2F-1 (track 2) orpLEX.E2F-5 (track 3) was assessed in yeast by assaying the activity ofpLexA-CYC1-lacZ. The data presented were derived from triplicatereadings.

FIG. 12

Pocket protein regulation E2F-5.

(a) Summary of reporter and effector constructs.

(b) The transcriptional activity of Gal-E2F-5 (track 2) was assessed inthe presence of wild-type pRb (track 3), pRbΔ22 (track 4), p107 (track5) and p107AS (track 6). For comparison, similar treatments wereperformed with pG4 (tracks 7 to 10). The data presented were derivedfrom triplicate readings.

FIG. 13

E2F-5 is a physiological DNA binding component of DRTF1/E2F in F9 ECcells.

Two types of antisera raised against distinct peptides from differentregions within E2F-5 were assessed for their effect on F9 EC cellDRTF1/E2F DNA binding activity. The anti-E2F-5 sera, anti-peptide 1(tracks 5 to 8) and anti-peptide 2 (track 9 to 12) were assessed in thepresence of either the homolgous (+; tracks 5, 7, 9 and 11) or anunrelated (−; tracks 6, 8, 10 and 12) peptide. For comparison, theeffect of anti-DP-1(24) (tracks 1 to 4) in the presence of either thehomologous (tracks 1 and 3) or an unrelated peptide (tracks 2 and 4) wasassessed. Each pair or tracks (+ together with −) represents treatmentwith a different preparation of antiserum. The DRTF1/E2F b/c DNA bindingcomplexes (La Thangue et al., 1990) are indicated.

FIG. 14

DNA binding properties of E2F-5.

E2F-5, E2F-1 and DP-1 were expressed as GST fusion proteins, purifiedand their DNA binding activity assayed by gel retardation. Either E2F-1or E2F-5 were assayed alone (tracks 1 and 2, and 5, 6, and 7respectively) or together with a constant concentration of DP-1 (tracks3 and 4, and 8,9 and 10). Track 11 shows the probe alone. The amount ofproteins added for E2F-1 was approximately 50 ng (tracks 1 and 3) or 100ng (tracks 2 and 4), for E2F-5 25 ng (tracks 5 and 8), 50 ng (tracks 6and 9) or 100 ng (tracks 7 and 10), and for DP-1 50 ng throughout.

FIG. 15

Levels of E2F-5 RNA.

The levels of E2F-5 RNA were compared to E2F-1 in the indicated celllines. The level of GAPDH RNA was assessed as an internal control.

It has been found that E2F-S is one of a family of related transcriptionfactor components. Members of this family are believed to interact withDP proteins to form a series of transcription factors. DP proteins (orpolypeptides) include DP-1, DP-2 and DP-3 although the first of thesewill usually be contemplated in preference to the others.

The invention in a first aspect provides a protein as shown in FIG. 1Aor 9A, homologues thereof, and fragments of the sequence and theirhomologues, which can function as a mammalian transcription factor. Inparticular, the invention provides a polypeptide (preferably insubstantially isolated form) comprising:

(a) E2F-5;

(b) the protein of FIG. 1A or 9A;

(c) a mutant, allelic variant or species homologue of (a) or (b);

(d) a protein at least 70% homologous to (a) or (b);

(e) a fragment of any one of (a) to (d) capable of forming a complexwith a DP protein, pRb, p107 and/or p130; or

(f) a fragment of any of (a) to (e) of at least 15 amino acids long.

All polypeptides within this definition are referred to below aspolypeptide(s) according to the invention.

The proteins pRb, p107, DP proteins and p130 are referred to herein ascomplexing proteins or “complexors” as they may form a complex with theproteins of the invention. Under certain conditions E2F-5 may only bindweakly to pRb.

A polypeptide of the invention will be in substantially isolated form ifit is in a form in which it is free of other polypeptides with which itmay be associated in its natural environment (eg the body). It will beunderstood that the polypeptide may be mixed with carriers or diluentswhich will not interfere with the intended purpose of the polypeptideand yet still be regarded as substantially isolated.

The polypeptide of the invention may also be in a substantially purifiedform, in which case it will generally comprise the polypeptide in apreparation in which more than 90%, eg. 95%, 98% or 99% of thepolypeptide in the preparation is a polypeptide of the invention.

Mutant polypeptides will possess one or more mutations which areadditions, deletions, or substitutions of amino acid residues.Preferably the mutations will not affect, or substantially affect, thestructure and/or function and/or properties of the polypeptide. Thus,mutants will suitably possess the ability to be able to complex with DPproteins, pRb, p107 and/or p130. Mutants can either be naturallyoccurring (that is to say, purified or isolated from a natural source)or synthetic (for example, by performing site-directed mutagenesis onthe encoding DNA). It will thus be apparent that polypeptides of theinvention can be either naturally occurring or recombinant (that is tosay prepared using genetic engineering techniques).

An allelic variant will be a variant which will occur naturally in ahuman or in an, eg. murine, animal and which will function to regulategene expression in a substantially similar manner to the protein in FIG.1A or 9A.

Similarly, a species homologue of the protein will be the equivalentprotein which occurs naturally in another species, and which performsthe equivalent function in that species to the protein of FIG. 1A or 9A.Within any one species, a homologue may exist as several allelicvariants, and these will all be considered homologues of the protein.Allelic variants and species homologues can be obtained by following theprocedures described herein for the production of the protein of FIG. 1Aor 9A and performing such procedures on a suitable cell source, eg fromhuman or a rodent, carrying an allelic variant or another species. Sincethe protein may be evolutionarily conserved it will also be possible touse a polynucleotide of the invention to probe libraries made fromhuman, rodent or other cells in order to obtain clones encoding theallelic or species variants. The clones can be manipulated byconventional techniques to identify a polypeptide of the invention whichcan then be produced by recombinant or synthetic techniques known perse. Preferred species homologues include mammalian or amphibian specieshomologues.

A protein at least 70% homologous to that in FIG. 1A or 9A is includedin the invention, as are proteins at least 80 or 90% and more preferablyat least 95% homologous to the protein shown in these Figures. This willgenerally be over a region of at least 20, preferably at least 30, forinstance at least 40, 60 or 100 or more contiguous amino acids. Methodsof measuring protein homology are well known in the art and it will beunderstood by those of skill in the art that in the present context.Homology is usually calculated on the basis of amino acid identity(sometimes referred to as “hard homology”).

Generally, fragments of the polypeptide in FIG. 1A or 9A or its allelicvariants or species homologues thereof capable of forming a complex withthe complexors will be at least 10, preferably at least 15, for exampleat least 20, 25, 30, 40, 50 or 60 amino acids in length.

It will be possible to determine whether fragments form a complex withthe complex of proteins by providing the complexor protein and thefragment under conditions in which they normally form a trans-activatingtranscription factor, and determining whether or not a complex hasformed. The determination may be made by, for example, measuring theability of the complex to bind an E2F binding site in vitro, oralternatively determining the molecular weight of the putative complexby methods such as SDS-PAGE.

Preferred fragments include those which are capable of forming atrans-activation complex with DP-1 or other complexors. The examplesherein describe a number of methods to analyse the function of theprotein and these may be adapted to assess whether or not a polypeptideis capable of forming a trans-activation complex with the DP-1 protein.For example, the polypeptide can be added to the complexor in thepresence of a reporter gene construct adapted to be activated by theDP-1/E2F-5 complex (for example, see FIG. 10 of WO-A-94/10307 in thename of the Medical Research Council). Such an experiment can determinewhether the polypeptide fragment has the necessary activity.

A polypeptide of the invention may be labelled with a revealing ordetectable label. The (revealing) label may be any suitable label whichallows the polypeptide to be detected. Suitable labels includeradioisotopes, e.g. ¹²⁵I, enzymes, antibodies and linkers such asbiotin. Labelled polypeptides of the invention may be used in diagnosticprocedures such as immunoassays in order to determine the amount ofE2F-5 protein in a sample.

A polypeptide or labelled polypeptide according to the invention mayalso be fixed to a solid phase, for example the wall of an immunoassaydish.

A second aspect of the invention relates to a polynucleotide whichcomprises:

(a) a sequence of nucleotides shown in FIG. 1A or 9A;

(b) a sequence complementary to (a);

(c) a sequence capable of selectively hybridising to a sequence ineither (a) or (b);

(d) a sequence encoding a polypeptide as defined in the first aspect; or

(e) a fragment of any of the sequences in (a) to (d).

The present invention thus provides a polynucleotide, suitably insubstantially isolated or purified form, which comprises a contiguoussequence of nucleotides which is capable of selectively hybridizing tothe sequence of FIG. 1A or 9A or to a complementary sequence.

Polynucleotides of the invention include a DNA sequence in FIG. 1A or 9Aand fragments thereof capable of selectively hybridizing to the sequenceof FIG. 1A or 9A. A further embodiment of the invention provides a DNAcoding for the protein in FIG. 1A or 9A or a fragment thereof.

The polynucleotide may also comprise RNA. It may also be apolynucleotide which includes within it synthetic or modifiednucleotides. A number of different types of modification tooligonucleotides are known in the art. These include methylphosphonateand phosphorothionate backbones, addition of acridine or polylysinechains at the 3′ and/or 5′ ends of the molecule. For the purposes of thepresent invention, it is to be understood that the oligonucleotidesdescribed herein may be modified by any method available in the art.Such modifications may be carried out in order to enhance the in vivoactivity or lifespan of oligonucleotides of the invention used inmethods of therapy.

A polynucleotide capable of selectively hybridizing to the DNA of FIG.1A or 9A will be generally at least 70%, preferably at least 80 or 90%and optimally at least 95% homologous to the DNA of FIG. 1A or 9A over aregion of at least 20, preferably at least 30, for instance at least 40,60 or 100 or more contiguous nucleotides. These polynucleotides arewithin the invention.

A polynucleotide of the invention will be in substantially isolated formif it is in a form in which it is free of other polynucleotides withwhich it may be associated in its natural environment (usually thebody). It will be understood that the polynucleotide may be mixed withcarriers or diluents which will not interfere with the intended purposeof the polynucleotide and it may still be regarded as substantiallyisolated.

A polynucleotide according to the invention may be used to produce aprimer, e.g. a PCR primer, a probe e.g. labelled with a revealing ordetectable label by conventional means using radioactive ornon-radioactive labels, or the polynucleotide may be cloned into avector. Such primers, probes and other fragments of the DNA of FIG. 1Aor 9A will be at least 15, preferably at least 20, for example at least25, 30 or 40 nucleotides in length, and are also encompassed within theinvention.

Polynucleotides, such as a DNA polynucleotides according to theinvention may be produced recombinantly, synthetically, or by any meansavailable to those of skill in the art. It may be also cloned byreference to the techniques disclosed herein.

The invention includes a double stranded polynucleotide comprising apolynucleotide according to the invention and its complement.

A third aspect of the invention relates to an (eg. expression) vectorsuitable for the replication and expression of a polynucleotide, inparticular a DNA or RNA polynucleotide, according to the invention. Thevectors may be, for example, plasmid, virus or phage vectors providedwith an origin of replication, optionally a promoter for the expressionof the polynucleotide and optionally a regulator of the promoter. Thevector may contain one or more selectable marker genes, for example anampicillin resistance gene in the case of a bacterial plasmid or aneomycin resistance gene for a mammalian vector. The vector may be usedin vitro, for example for the production of RNA or used to transfect ortransform a host cell. The vector may also be adapted to be used invivo, for example in a method of gene therapy.

Vectors of the third aspect are preferably recombinant replicablevectors. The vector may thus be used to replicate the DNA. Preferably,the DNA in the vector is operably linked to a control sequence which iscapable of providing for the expression of the coding sequence by a hostcell. The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under condition compatible with the controlsequences. Such vectors may be transformed or transfected into asuitable host cell to provide for expression of a polypeptide of theinvention.

A fourth aspect of the invention thus relates to host cells transformedor transfected with the vectors of the third aspect. This may allow forthe replication and expression of a polynucleotide according to theinvention, including the sequence of FIG. 1A or 9A or the open readingframe thereof. The cells will be chosen to be compatible with the vectorand may for example be bacterial, yeast, insect or mammalian.

A polynucleotide according to the invention may also be inserted intothe vectors described above in an antisense orientation in order toprovide for the production of antisense RNA. Antisense RNA or otherantisense polynucleotides may also be produced by synthetic means. Suchantisense polynucleotides may be used in a method of controlling thelevels of the E2F-5 protein in a cell. Such a method may includeintroducing into the cell the antisense polynucleotide in an amounteffective to inhibit or reduce the level of translation of the E2F-5mRNA into protein. The cell may be a cell which is proliferating in anuncontrolled manner such as a tumour cell.

Thus, in a fifth aspect the invention provides a process for preparing apolypeptide according to the invention which comprises cultivating ahost cell transformed or transfected with an (expression) vector of thethird aspect under conditions providing for expression (by the vector)of a coding sequence encoding the polypeptide, and recovering theexpressed polypeptide.

The invention in a sixth aspect also provides (monoclonal or polyclonal)antibodies specific for a polypeptide according to the invention.Antibodies of the invention include fragments, thereof as well asmutants that retain the antibody's binding activity. The inventionfurther provides a process for the production of monoclonal orpolyclonal antibodies to a polypeptide of the invention. Monoclonalantibodies may be prepared by conventional hybridoma technology usingthe proteins or peptide fragments thereof as an immunogen. Polyclonalantibodies may also be prepared by conventional means which compriseinoculating a host animal, for example a rat or a rabbit, with apolypeptide of the invention and recovering immune serum.

Fragments of monoclonal antibodies which can retain their antigenbinding activity, such Fv, F(ab′) and F(ab₂)′ fragments are included inthis aspect of the invention. In addition, monoclonal antibodiesaccording to the invention may be analyzed (eg. by DNA sequence analysisof the genes expressing such antibodies) and humanized antibody withcomplementarity determining regions of an antibody according to theinvention may be made, for example in accordance with the methodsdisclosed in EP-A-0239400 (Winter).

The present invention further provides compositions comprising theantibody or fragment thereof of the invention together with a carrier ordiluent. Such compositions include pharmaceutical compositions in whichcase the carrier or diluent will be pharmaceutically acceptable.

Polypeptides of the invention can be present in compositions togetherwith a carrier or diluent. These compositions include pharmaceuticalcompositions where the carrier or diluent will be pharmaceuticallyacceptable.

Pharmaceutically acceptable carriers or diluents include those used informulations suitable for oral, rectal, nasal, topical (including buccaland sublingual), vaginal or parenteral (including subcutaneous,intramuscular, intravenous, intradermal, intrathecal and epidural)administration. The formulations may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy. Such methods include the step of bringing intoassociation the active ingredient with the carrier which constitutes oneor more accessory ingredients. In general the formulations are preparedby uniformly and intimately bringing into association the activeingredient with liquid carriers or finely divided solid carriers orboth, and then, if necessary, shaping the product.

For example, formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants. buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the polypeptide to blood componentsor one or more organs.

Polypeptides according to the invention, antibodies or fragments thereofto polypeptides according to the invention and the above-mentionedcompositions may be used for the treatment, regulation or diagnosis ofconditions, including proliferative diseases, in a mammal including man.Such conditions include those associated with abnormal (eg at anunusually high or low level) and/or aberrant (eg due to a mutation inthe gene sequence) expression of one or more transcription factors suchas the DP or E2F proteins or related family members. The conditions alsoinclude those which are brought about by abnormal expression of a genewhose gene product is regulated by the protein of FIG. 1A or 9A.Treatment or regulation of conditions with the above-mentioned peptides,antibodies, fragments thereof and compositions etc. will usually involveadministering to a recipient in need of such treatment an effectiveamount of a polypeptide, antibody, fragment thereof or composition, asappropriate.

The invention also provides antibodies, and fragments thereof, targetedto this region in order to inhibit the activation of transcriptionfactors via the disruption of the formation of the E2F-5-DP proteincomplex.

The present invention further provides a method of performing animmunoassay for detecting the presence or absence of a polypeptide ofthe invention in a sample, the method comprising:

(a) providing an antibody according to the invention;

(b) incubating the sample with the antibody under conditions that allowfor the formation of an antibody-antigen complex; and

(c) detecting, if present, the antibody-antigen complex.

In another aspect, the invention provides a novel assay for identifyingputative chemotherapeutic agents for the treatment of proliferative orviral disease which comprises bringing into contact a DP protein or aderivative thereof, a polypeptide of the invention and a putativechemotherapeutic agent, and measuring the degree of inhibition offormation of the DP/E2F-5 protein complex caused by the agent. It maynot be necessary to use complete DP-1 and/or E2F-5 protein in the assay,as long as sufficient of each protein is provided such that under theconditions of the assay in the absence of agent, they form aheterodimer.

The cloning and sequencing of DP-1 (and E2F 1,2 and 3) are known in theart and methods for the recombinant expression and preparation ofantibodies to DP-1 can be found in WO-A-94/10307.

Thus, the invention provides a screening method for identifying putativechemotherapeutic agents for the treatment of proliferative disease whichcomprises:

(A) bringing into contact:

(i) a DP polypeptide;

(ii) a polypeptide of the first aspect, and

(iii) a putative chemotherapeutic agent;

under conditions in which the components (i) and (ii) in the absence of(iii) form a complex; and

(B) measuring the extent to which component (iii) is able to disruptsaid complex.

In the assay, any one or more of the three components may be labelled,eg with a radioactive or calorimetric label, to allow measurement of theresult of the assay. Putative chemotherapeutic agents include peptidesof the invention.

Variants, homologues and fragments of DP proteins are defined in acorresponding manner to the variants, homologues and fragments of theE2F-5 protein.

The complex of (i) and (ii) may be measured, for example, by its abilityto bind an E2F DNA binding site in vitro. Alternatively, the assay maybe an in vivo assay in which the ability of the complex to activate apromoter comprising an E2F binding site linked to a reporter gene ismeasured. The in vivo assay may be performed for example by reference tothe examples which show such an assay in yeast, insect, amphibian ormammalian cells.

Candidate therapeutic agents which may be measured by the assay includenot only polypeptides of the first aspect, but in particular fragmentsof 10 or more amino acids of:

(a) the protein of FIG. 1A or 9A;

(b) an allelic variant or species homologue thereof; or

(c) a protein at least 70% homologous to (a).

Vectors carrying a polynucleotide according to the invention or anucleic acid encoding a polypeptide according to the invention may beused in a method of gene therapy. Such gene therapy may be used to treatuncontrolled proliferation of cells, for example a tumour cell. Methodsof gene therapy include delivering to a cell in a patient in need oftreatment an effective amount of a vector capable of expressing in thecell either an antisense polynucleotide of the invention in order toinhibit or reduce the translation of E2F-5 mRNA into E2F-5 protein or apolypeptide which interferes with the binding of E2F-5 to a DP proteinor a related family member.

The vector is suitably a viral vector. The viral vector may be anysuitable vector available in the art for targeting tumour cells. Forexample, Huber et al (Proc. Natl. Acac. Sci. USA (1991) 88, 8039) reportthe use of amphotrophic retroviruses for the transformation of hepatoma,breast, colon or skin cells. Culver et al (Science (1992) 256;1550-1552) also describe the use of retroviral vectors in virus-directedenzyme prodrug therapy, as do Ram et al (Cancer Research (1993) 53;83-88). Englehardt et al (Nature Genetics (1993) 4; 27-34 describe theuse of adenovirus based vectors in the delivery of the cystic fibrosistransmembrane conductance product (CFTR) into cells.

The invention contemplates a number of assays. Broadly, these can beclassified as follows.

1. Conducting an assay to find an inhibitor of E2F-5 trans-activation(that is to say, inhibition of activation of transcription). Thisinhibitor may therefore inhibit binding of E2F-5 to DNA (usually at theE2F binding site). Potentially suitable inhibitors are proteins, and mayhave a similar or same effect as p107. Thus suitable inhibitorymolecules may comprise fragments, mutants, allelic variants, or specieshomologues of p107 in the same manner as defined for proteins of thefirst aspect.

2. Assaying for inhibitors of (hetero)dimerisation. Such inhibitors mayprevent dimerisation of E2F-5 (or a polypeptide of the first aspect)with a complexor, for example a DP protein, such as DP-1. Of course theinhibitor can be a fragment, mutant, allelic variant or specieshomologue of a DP protein in a similar manner as defined for theproteins of the first aspect.

3. A third category of assay is to find inhibitors of phosphorylation.It is thought that E2F-5 (and other proteins of the first aspect) mightbe activated by phosphorylation. Therefore, an inhibitor ofphosphorylation is likely to inhibit E2F-5 trans-activation properties(and may therefore, ultimately have the same effect as the inhibitorsfound in either of the two previous assays). Phosphorylation is by cdk'sand so an inhibitor of this phosphorylation is one that is contemplatedby such assays.

The invention contemplates a number of therapeutic uses. For example,gene therapy using a nucleic acid a sequence that is antisense to E2F-5.Molecules that can bind to a DP-1 protein and thereby form an inactivecomplex with the DP protein are additionally contemplated. Suitablemolecules include those of the first aspect apart from E2F-5 itself.Such molecules may be mutants of E2F-5, and are often referred to asdominant negative molecules in the art.

The invention contemplates the treatment or prophylaxis of diseases thatare based on the uncontrolled proliferation of cells, or whereuncontrolled proliferation is an important or essential pathologicalaspect of the disease. This includes cancer, viral disease, selfproliferation itself as well as auto immune diseases such psoriasis. Onemay also wish to temporarily inhibit the growth of dividing cells, forexample hematopoietic stem cells and/or bone marrow cells. In theseaspects one is generally seeking to prevent, inhibit or interfere withthe activity of E2F-5.

In contrast some diseases and conditions can be treated by increasingE2F-5 expression, for example by promoting or inducing overexpression.This preferably results in apoptosis, sometimes known as programmed celldeath. Overexpression of the E2F-5 protein can result in death of thecell, and therefore this aspect can also be used in the treatment ofcancer. One aim is therefore to increase the activity of E2F-5. Similaruses are known for E2F-1 (Qin et al, PNAS USA 91 (in press)).

It should be borne in mind that the E2F-5 gene might be mutated intumour cells. In that event, the mutated gene could be used in diagnosisof a condition resulting from the mutation. It also lends itself totreatment via the mutated gene.

The following two Examples describe the isolation and characterizationof the novel protein and DNA of the invention from human and murinesources, respectively. However, other e.g. mammalian sources are withinthe scope of the present invention and other mammalian homologues of theprotein may be isolated in an analogous manner. The Examples arepresented here by way of illustration and are not to be construed aslimiting on the invention.

EXAMPLE 1 SUMMARY

E2F DNA binding sites are found in a number of genes whose expression istightly regulated during the cell cycle. The activity of E2Ftranscription factors is regulated by association with specificrepressor molecules that can bind and inhibit the E2F transactivationdomain. For E2F-1, 2 and 3 the repressor is the product of theretinoblastoma gene, pRb. E2F-4 interacts with pRb-related p107 and notwith pRb itself. Recently, a cDNA encoding a third member of theretinoblastoma gene family, p130, was isolated. p130 also interacts withE2F DNA binding activity, primarily in the G₀ phase of cell cycle. Wereport here the cloning of a fifth member of the E2F gene family. Thehuman E2F-5 cDNA encodes a 346-amino acid protein with a predictedmolecular mass of 38 kDa. E2F-5 is more closely related to E2F-4 (78%similarity) than to E2F-1 (57% similarity). E2F-5 resembles the otherE2Fs in that it binds to a consensus E2F site in a cooperative fashionwith DP-1. Using a specific E2F-5 antiserum, we show that underphysiological conditions E2F-5 interacts preferentially with p130.

Introduction

E2F is the name given to a group of heterodimeric transcription factorsthat are composed of an E2F-like and a DP-like subunit [27]. E2F DNAbinding sites are present in the promoters of a number of genes whoseexpression is regulated during the cell cycle and evidence exists toindicate that the presence of these E2F sites contributes to the cellcycle-regulated expression of these genes [13, 28, 38].

E2F DNA binding activity has been found in complex with theretinoblastoma protein (pRb) and the pRb-related p107 and p130 [6, 10,29, 37]. This group of proteins shares a conserved motif, the “pocket”,that is involved in binding to both cellular and viral proteins. Forthis reason, the group of pRb-like proteins is collectively known as thepocket protein family. Complexes between E2F and the various pocketproteins are likely to have different functions in cell cycle regulationas their appearance differs during the cell cycle. E2F in complex withpRb is found mostly in G₁ phase of the cell cycle [5-7, 11]. Incontrast, complexes between p107 and E2F persist during the cell cycle,but their composition is variable. In G₁, apart from E2F and p107,cyclin E and cdk2 are present. In S phase, cyclin E is replaced bycyclin A in the E2F/p107 complex [29, 37]. The functional significanceof the presence of these cyclin/cdk complexes in the p107/E2F complex isnot clear at present. In quiescent cells, a complex between E2F and p130is the most prominent E2F DNA binding species. This complex disappearsas cells emerge from quiescence, suggesting a role for p130-interactingE2F activity in cell cycle entry [10].

The ability of E2F to activate transcription is regulated by complexformation with the pocket proteins. Complex formation between E2F andpRb is subject to regulation by phosphorylation. Only thehypophosphorylated species of pRb interact with E2F, indicating. thatthe phosphorylation of pRb by cyclin/cdk complexes controls theinteraction between E2F and pRb during the cell cycle [5-7, 11].

The crucial role of E2F transcription factors in cell cycle regulationis emphasized by the finding that enforced expression of E2F DNA bindingactivity causes cells to progress from G₁ into S and G₂/M phases of thecell cycle [3] and E2F can stimulate quiescent cells to initiate DNAsynthesis [23]. Importantly, over-expression of E2F, together with anactivated ras oncogene can cause oncogenic transformation of primaryrodent fibroblasts [3].

To date four different E2F-like polypeptides have been isolated. E2F-1,2 and 3 are found only in complex with pRb, whereas E2F-4 interactspreferentially with p107 [3, 15, 19, 22, 24, 30, 36]. How complexformation between E2F and p107 and E2F and p130 is regulated iscurrently not known. To begin to address the regulation of the E2F/p107complex and the E2F/p130 complex, we have searched for additionalmembers of the E2F gene family. We report here the cloning of a fifthmember of the E2F gene family that interacts preferentially with p130.

Materials and Methods

Yeast Two-hybrid Screen

Yeast strain Y190 [17], containing the ‘bait’ plasmid pPC97-p107, wastransformed with a day 14.5 CD1 mouse embryo library [8] using thelithium acetate method [34]. Two million transformants were selected forgrowth on plates lacking histidine and supplemented with 25 mM3-aminotriazole and subsequently analyzed for β-galactosidase activityas previously described [12]. cDNAs library plasmids derived from doublypositive yeast colonies were tested for target specificity byre-transformation with different Gal4-DBD fusion plasmids: pPC97-p107,pPC97-bmi and pPC97 without insert. The partial mouse E2F-5 cDNA wasused to screen additional human cDNA libraries. The full length humanE2F-5 cDNA described here was isolated from the T84 colon carcinomalibrary (Stratagene).

Plasmids

pPC97-p107 was generated by cloning the pocket region of p107 (aminoacids 240-816) in frame with the Gal4 DNA binding domain (amino acids1-147) of pPC97 [8]. pGST-E2F-5 (A) and (B ) were constructed by cloninga fragment of human E2F-5 cDNA encoding amino acids 89-200 (A) or aminoacids 89-346 (B) in pGEX-2T. For transfection experiments the followingplasmids were used: pSG-Gal4-E2F-1 contains amino acids 284-437 of humanE2F-1 [19]. pJ3-Gal4-E2F-4 and pJ3-Gal4-E2F-5 were obtained by cloning afragment of the human cDNA of E2F-4 (encoding amino acids 276-412) orE2F-5 (encoding amino acids 222-346) in frame with the DNA bindingdomain of Gal4 in pJ3Ω[33]. pJ3-E2F-5 was constructed by cloning thefull length human E2F-5 cDNA (lacking the last 184 nucleotides of 3′ noncoding sequence) into the mammalian expression vector pJ3Ω. Thetranslation start codon of E2F-5 was preceded by the 10 amino acidepitope (HA) that is recognized by the monoclonal antibody 12CA5.pCMV-DP-1 , pCMV-pRb, pCMV-p107, pCMV-p107DE , PCMV-pRbΔ22 have beendescribed previously [20, 41].

Cell Lines

U2-OS and CAMA cells were cultured in Dulbecco's modified Eagle medium(DMEM) supplemented with 10% or 20% fetal calf serum, respectively.Transfections were performed using the calcium phosphate precipitationtechnique [39].

CAT Assays

U2-OS cells were transiently transfected with the expression vectors asindicated together with 5 μg (Gal4)₅-CAT [25] or 2 μg E2F₄-CAT [20], 0.2μg RSV-luciferase and herring sperm carrier DNA to a total amount of 20μg/10 cm plate. Cells were assayed for CAT and luciferase activity asdescribed previously [2, 3].

Northern Blot Analysis

For E2F-5 expression analysis, total cytoplasmic RNA was prepared from apanel of cell lines. 20 mg of total cellular RNA was electrophoresedthrough a 1% formaldehyde agarose gel as described [4], transferred tonitrocellulose and probed with a [³²P]-labeled partial human E2F-5 cDNA(nt. 666-1038). Subsequently, the same filter was probed with a ratα-tubulin cDNA to control for the amount of RNA loaded in each lane.

Immunological Reagents and Immunoprecipitations

To generate antibodies against E2F-5, GST-E2F-5 (A) and (B) (seeplasmids) proteins were made in E. coli and purified usinggluthatione-sepharose beads. Both proteins were injected in a rabbit inequal amounts. After three rounds of immunization polyclonal serum wasobtained. Monoclonal antibodies against E2F-1 (KH20), E2F-4 (RK13), pRb(XZ77) and p107 (SD-4 and 9) have been described previously [3, 20, 21,41]. The p130 (C20) rabbit polyclonal antiserum was obtained from SantaCruz Biotechnology Inc. CAMA cells and transfected U2-OS cells werelabeled and immunoprecipitated as described previously [3].

Gel Retardation Assays

Gel retardation assays for transiently transfected U-2 OS cells wereperformed as described previously [20] with minor modifications. 10 μgof whole cell extract was used in a binding buffer containing 20 mMHEPES (pH 7.4), 0.1 M KCl, 1 mM MgCl₂, 0.1 mM EDTA, 7% glycerol, 1 mMNaF and 1 μg sonicated salmon sperm DNA in 20 μl reaction volume with0.5 ng of [³²P]-labeled oligonucleotide specifying the consensus E2F DNAbinding site (Santa Cruz Biotechnology). DNA-protein complexes wereallowed to form during an incubation for 20 min. at RT. The reactionproducts were separated on a 3.5% polyacrylamide gel in 0.25×TBE at 90Vat RT for 2.5 hours. The gel was then dried and exposed to film.

Results

Isolation of p107 Binding Proteins

To identify cDNAs encoding polypeptides that interact with p107, a yeasttwo hybrid screen was performed [14]. Yeast strain Y190 [17], whichcontains two chromosomally located Gal4-inducible reporter genes: HIS3and LacZ [12], was co-transformed with the ‘bait’ plasmid containing thepocket region (amino acids 240-816) of p107 fused to the DNA bindingdomain (DBD) of Gal4 and a day 14.5 CD1 mouse embryo cDNA library inwhich each cDNA is individually fused to the transactivation domain ofGal4 [8]. A total of 2 million transformants were placed under selectionon plates lacking histidine. Eighty seven surving colonies were screenedfor expression of β-galactosidase. cDNA-containing plasmids were rescuedfrom sixteen doubly positive yeast colonies. The specificity ofp107-binding was confirmed by re-transformation with plasmids encodingother Gal4-DBD fusions. All sixteen hybrid proteins were found tointeract specifically with Gal4-p107. DNA sequence analysis showed thatthe sixteen cDNA library plasmids rescued from the yeasts were derivedfrom 10 different genes. Three cDNAs were derived from the same gene andshowed significant homology to the four known E2Fs. Because of this wenamed the protein encoded by this cDNA E2F-5.

The partial mouse E2F-5 cDNA was then used to obtain a full length humancDNA clone by screening a human colon carcinoma cDNA library. Thelongest cDNA (2.1 kb) was sequenced and contained a 1038 bp open readingframe encoding a 346-amino acid protein with a predicted molecular massof 38 kDa. FIG. 1A shows the E2F-5 cDNA sequence and the deduced aminoacid sequence.

E2F-5 is more closely related to E2F-4 (78% similarity) than to E2F-1(57% similarity). In comparison with E2F-1 and E2F4, three regions ofhomology can be observed in E2F-5. (FIG. 1B). The DNA binding domain(amino acids 43-115 of E2F-5) shares 93% similarity with the E2F-4 DNAbinding region, whereas the juxtaposed DP-1 dimerization domain is 81%similar between E2F-4 and E2F-5. Finally, the carboxyl terminal pocketprotein interaction domain of E2F-4 and 5 are 83% similar. E2F-4 andE2F-5 differ from E2F-1 in that both proteins lack the amino terminalmotif of E2F-1 that is involved in cyclin A binding. E2F-5 differs fromE2F in that it lacks the serine repeat region of E2F-4.

To analyze mRNA expression levels of E2F-5, a human E2F-5 cDNA was usedto probe a Northern blot containing total cytoplasmic RNA from a numberof human cell lines. The E2F-5 probe detected a low level of a single2.1 kb transcript in most cell lines. The human CAMA breast carcinomacell line expressed somewhat higher levels of E2F-5 (FIG. 2).

E2F-5 Contains a Carboxyl-terminal Transactivation Domain

E2F-1 and E2F-4 contain a carboxyl-terminal transactivation domain thatoverlaps with the pocket protein binding site [3, 18]. To test whetherE2F-5 also contains a transactivation domain, we fused the carboxylterminus of human E2F-5 to the DNA binding domain of Gal4 in themammalian expression vector pJ3Ω. U2-OS osteosarcoma cells weretransiently transfected with a CAT reporter gene harboring five upstreamGal4 sites or cotransfected with the reporter gene and Gal4-E2Fexpression vectors. FIG. 3 shows that cotransfection of the Gal4reporter plasmid with the Gal4-E2F-5 expression vectors resulted in a50-fold activation of the CAT reporter gene. Cotransfection withGal4-E2F-1 or Gal4-E2F-4 resulted in a two- to three-fold higheractivation of the CAT reporter gene (FIG. 3). We conclude that E2F-5contains a potent carboxyl terminal transactivation domain.

E2F-5 Requires DP-1 for DNA Binding

Both E2F-1 and E2F-4 require dimerization with DP-1 for efficient DNAbinding [1, 3, 20, 26]. To investigate whether E2F-5 can bind to aconsensus E2F DNA binding site and whether E2F-5 requires DP-1dimerization in order to bind DNA, we performed a transient transfectionexperiment. Human U2-OS osteosarcoma cells were transfected with a CATreporter plasmid in which a core promoter was linked to four upstreamE2F sites. FIG. 4 shows that the E2F-CAT reporter plasmid only has lowactivity when transfected alone in the osteosarcoma cells. Transfectionof DP-1 or E2F-5 expression vectors separately did not result inactivation of the E2F-CAT reporter (FIG. 4, tracks 2 and 6).Cotransfection of DP-1 and E2F-5 expression vectors resulted in a strongdose-dependent synergistic activation of the CAT reporter (FIG. 4,tracks 3-5). These data indicate that E2F-5 can bind the consensus E2Fsite and that DNA-binding is DP-1-dependent. Based on these results weconclude that E2F-5 is a genuine member of the E2F gene family.

E2F-5 Transactivation is Suppressed by Pocket Proteins

Transactivation of E2F-1 and E2F-4 is suppressed by pocket proteinbinding because the transactivation domain of these E2Fs overlaps withthe pocket protein interaction surface. To test the effect of pocketprotein expression on E2F-5 transactivation we used a transienttransfection assay. Since E2F-1 and E2F-4 both require DP-1 dimerizationfor efficient binding to their respective pocket proteins [3, 20], wemeasured the effect of pocket protein expression on E2F-5 plus DP-1activated transcription. U2-OS cells were transfected with the E2F-CATreporter plasmid together with E2F-5 and DP-1. FIG. 5 (track 3) showsthat cotransfection of E2F-5 and DP-1 resulted in a greater than100-fold activation of the E2F-CAT reporter gene. E2F-5-stimulatedtranscription was inhibited by cotransfection with pRb, p107 and p130expression vectors in a dose-dependent fashion. Mutants of pRb (pRbΔ22)and p107 (p107DE) that lack an intact pocket domain were unable tosuppress E2F-5 transactivation (FIG. 5, tracks 6 and 9). Significantly,these mutant forms of pRb and p107 also lack growth inhibitory activity[41]. Thus, although this experiment did not allow for an unambiguousidentification of the preferred binding partner of E2F-5, it didindicate that E2F-5 transactivation is inhibited by pocket proteinbinding and that a close correlation exists between the ability of pRband p107 to cause a growth arrest and their ability to inhibit E2F-5transactivation. It is important to point out that the U2-OS cells usedin this experiment are insensitive to a pRb-or p107-induced growtharrest [41]. The observed effects on E2F-5 transactivation are thereforeunlikely to be due to non-specific cell cycle effects of pRb or p107.

E2F-5 Interacts Preferentially with p130 in a Band Shift Assay.

To further investigate the specificity of pocket protein binding byE2F-5, we performed an electrophoresis mobility shift assay (EMSA).U2-OS cells were transiently transfected with DP-1 and E2F-5 expressionvectors with or without pRb, p107 or p130 expression vectors. Two daysafter transfection, whole cell extracts were prepared from transfectedcells and incubated with a [³²P]-labeled oligonucleotide that specifiesa consensus E2F site. DNA-protein complexes were separated on apolyacrylamide gel and visualized by radiography. FIG. 6 shows thattransfection of E2F-5 and DP-1 expression vectors leads to theappearance of a novel complex that was not observed in themock-transfected cells (FIG. 6, compare lanes 1 and 2). This complexcould be supershifted by cotransfection of p130 expression vector, butnot by p107 or pRb expression vectors (FIG. 6, lanes 3-5). These datasuggest that of the three pocket proteins tested, p130 has the highestaffinity for the E2F-5/DP-1 heterodimer.

E2F-5 Interacts Preferentially with p130 in vivo.

Under physiological conditions, E2F-1 binds preferentially to pRb andE2F-4 to p107 [3, 15, 19, 24]. In transient transfection experimentshowever, both E2F-1 and E2F-4-activated gene expression can besuppressed by both pRb and p107 [3, 40]. This loss of specificity isprobably caused by the transient over-expression of these proteins. Toaddress which of the three members of the retinoblastoma protein familyinteracts with E2F-5 under physiological conditions, we generated apolyclonal rabbit antiserum against human E2F-5. Initialimmunoprecipitation experiments using in vitro transcribed andtranslated E2F-1, E2F-4 and E2F-5 indicated that the polyclonal E2F-5serum specifically recognized E2F-5 (data not shown). The E2F-5antiserum was then used in a sequential immunoprecipitation experiment.CAMA breast carcinoma cells were metabolically labeled with[³²p]-orthophosphate and non-ionic detergent lysates were prepared.These lysates were subjected to immunoprecipitation with pRb-specificantibody, p107 antibody or p130-specific antiserum. Proteins that wereco-immunoprecipitated with pRb, p107 or p130 were released by boiling inSDS-containing buffer, diluted, and re-immunoprecipitated withE2F-5-specific antiserum. FIG. 6 panel B shows that a protein of 47 kDacould be specifically re-immunoprecipitated with E2F-5 antiserum fromthe p130 immunoprecipitate, but not from pRb or p107 immunoprecipitates.This 47 kDa protein comigrates on SDS polyacrylamide gels withtransiently transfected E2F-5 (data not shown). As a control we verifiedwhether pRb and p107 immunoprecipitates contained their respective E2Fs.FIG. 6, panel C shows that pRb did indeed coimmunoprecipitate E2F-1 andp107 brought down E2F-4. Taken together these data indicate that E2F-5preferentially interacts with p130 in vivo.

Discussion

We report here the isolation of a fifth member of the E2F gene family.E2F-5 has all the hallmarks of a genuine E2F family member: it containsa highly conserved DNA binding domain, a DP-1 dimerization domain and acarboxyl terminal transactivation domain. Furthermore, E2F-5 binds aconsensus E2F DNA binding site in a cooperative fashion with DP-1 andcan activate the expression of an E2F site-containing reporter gene.

We performed three types of experiments to address with which of thethree pocket proteins E2F-5 interacts preferentially in vivo. Intransient transfection experiments, E2F-5 transactivation could besuppressed by all three members of the retinoblastoma protein family,pRb, p107 and p130. In this respect E2F-5 resembles E2F-1 and E2F4,since both E2F-1 and E2F-4 transactivation can be inhibited in transienttransfection assays by pRb as well as p107 [3, 40]. This apparent lackof specificity in a transient transfection assay is probably the resultof the high transient expression levels of both the E2F and the pocketproteins in the transiently transfected cells. The relatively low levelof inhibition of E2F-5 transactivation by p130 in the transienttransfection experiment (FIG. 5) is the result of the low level of p130expression since in transiently transfected cells, p107 was found to beexpressed at a 10-fold higher level as compared to p130 (data notshown). Two additional experiments were performed to address pocketprotein specificity of E2F-5. In the first experiment, cells weretransiently transfected with E2F-5 and DP-1 expression vectors in thepresence or absence of expression vectors for all three pocket proteins.Subsequently, band shift assays were performed using extracts from thetransfected cells with an oligonucleotide specifying a consensus E2Fbinding site. Only cotransfection of p130 could cause a supershift ofthe E2F-5/DP-1 complex (FIG. 6). In the band shift experiment, onlycomplexes between pocket proteins and E2F-5 that are stable forprolonged periods of time are detected as E2F/pocket protein“supershifted” complexes. Thus, even though p130 was expressed at alower level than p107, the complex between E2F-5 and p130 was morestable than the p107/E2F-5 complex (FIG. 6). In a similar experiment, wewere able to “supershift” an E2F-4 DNA binding complex with p107, butnot with pRb (R.L.B and R.B, unpublished data). This result suggeststhat mobility shift experiments can be potentially useful to addresspocket protein specificity of E2Fs. Consistent with the results of themobility shift assay, we found that in non-transfected metabolicallylabeled CAMA breast carcinoma cells, E2F-5 could beco-immunoprecipitated with p130, and not with p107 or pRb (FIG. 7).Taken together, our data indicate that under physiological conditions,E2F-5 preferentially associates with p130.

The finding that E2F-5 interacted with p130 but not with p107 wassomewhat unexpected because p130 and p107 are structurally closelyrelated and indeed p107 and p130 share the ability to bind cyclins A andE [16, 32, 41]. On the other hand, p107 and p130 differ in their abilityto interact with D type cyclins in vivo as only p107, and not p130,co-immunoprecipitates with anti D type cyclin antibodies [32].Importantly, the appearance of the p130/E2F and p107/E2F complexesdiffers in the cell cycle [9, 10, 29, 35, 37]. This suggests that p107and p130 have distinct functions during the cell cycle. The preferentialbinding of E2F-5 by p130 is consistent with such a distinct role forp130 in cell cycle regulation.

Our finding that E2F-5 can bind to a consensus E2F site by no meansrules out the possibility that E2F-5 interacts with a discrete subset ofE2F sites in vivo that is distinct from the E2F sites that are bound bythe other members of the E2F gene family. Consistent with such a bindingsite preference of the different E2Fs is the finding that the E2F sitesthat are present in the thymidine kinase gene promoter and in the b-mybpromoter interact preferentially with E2F/p107 complexes [28, 31]. Sincecomplexes between E2F and p130 are found mostly in quiescent cells anddisappear quickly after cells emerge from quiescence, it is likely thatE2F-5-responsive genes are involved in the early responses of restingcells to growth factor stimulation [10]. The availability of thep130-interacting E2F-5, should allow us to identify E2F-5-responsivegenes.

Acknowledgments

We thank P. Chevray for the gift of the mouse embryo cDNA library andyeast expression vectors, S. Elledge for yeast strain Y1090, M. Alkemafor the Gal4-bmi yeast expression vector, G. Hannon for the gift of thep130 expression vector, A. Bes-Gennissen for the gift of human cell lineRNA and Y. Ramos for preparing the Northern blot. This work wassupported by a grant from the Netherlands Organization for ScientificResearch (NWO).

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EXAMPLE 2 Summary

The transcription factor DRTF1/E2F is implicated in the control ofcellular proliferation due to its interaction with key regulators ofcell cycle progression, such as the retinoblastoma tumour suppressorgene product and related pocket proteins, cyclins and cyclin-dependentkinases. DRTF1/E2F DNA binding activity arises when a member of twodistinct families of proteins, DP and E2F, interact as DP/E2Fheterodimers. Here, we report the isolation and characterisation of anew member of the E2F family of proteins, called E2F-5. E2F-5 wasisolated through a yeast two hybrid assay in which a 14.5 d.p.c. mouseembryo library was screened for molecules capable of binding to murineDP-1, but also interacts with all known members of the DP family ofproteins. E2F-5 exists as a physiological heterodimer with DP-1 in thegeneric DRTF1/E2F DNA binding activity present in mammalian cellextracts, an interaction which results in co-operative DNA bindingactivity and transcriptional activation through the E2F site. A potenttranscriptional activation domain, which functions in both yeast andmammalian cells and resides in the C-terminal region of E2F-5, andexpression of the pRb-related protein p107, rather than pRb, inactivatesthe transcriptional activity of E2F-5. Comparison of the sequence ofE2F-5 with other members of the family indicates that E2F-5 shows agreater level of similarity with E2F-4 than to E2F-1, -2 and -3. Thestructural and functional similarity of E2F-5 and E2F-4 defines asubfamily of E2F proteins.

Introduction

Considerable evidence suggests that the cellular transcription factorDRTF1/E2F is involved in co-ordinating transcription with cell cycleprogression. For example, DRTF1/E2F appears to be one of the principaltargets through which the retinoblastoma tumour suppressor gene product(pRb) exerts its negative effects on cellular proliferation (La Thangue,1994). Thus, by regulating the transcriptional activity of DRTF1/E2F andhence the activity of target genes, many of which encode proteinsrequired for cell cycle progression (Nevins, 1992), pRb is able toinfluence progression through the early cell cycle. Natural mutations inRb, which occur in human tumour cells, encode proteins which fail tobind to DRTF1/E2F (Bandara et al., 1992; Heibert et al., 1992; Zamanianand La Thangue. 1992), underscoring the correlation betweende-regulating DRTF1/E2F and aberrant cell growth. Furthermore, thetransforming activity of viral oncoproteins, such as adenovirus E1a.human papilloma virus E7 and SV40 large T antigen, correlates with theirability to de-regulate DRTF1/E2F through the sequestration of pRb andrelated proteins (Nevins, 1992), providing further support for thisview.

Other molecules which play a central role in the cell cycle alsointeract with DRTF1/E2F. Cyclins A and E, together with the catalyticsubunit cdk2, bind to DRTF1/E2F either directly by contacting the DNAbinding components in the transcription factor (Krek et al., 1994) orindirectly through contacts which occur in the spacer region of thepRb-related pocket proteins p107 or p130 (Lees et al., 1992; Cobrinik etal., 1993). Although the role of the cyclin-cdk complex which associateswith p107 and p130 has yet to be resolved, the direct interactionbetween the cyclin A/cdk2 kinase complex and DRTF1/E2F has been shown toaffect its DNA binding activity (Krek et al., 1994).

The molecular composition of DRTF1/E2F is beginning to be uncovered. Itis now clear that the generic DNA binding activity DRTF1/E2F arises whenmembers of two distinct families of proteins interact as DP/E2Fheterodimers (La Thangue, 1994), the prototype molecules of each familybeing DP-1 (Girling et al., 1993) and E2F-1 (Helin et al., 1992; Kaelinet al., 1992; Shan et al., 1992). A small region of similarity betweenboth proteins allows them to interact as a heterodimer (Bandara et al.,1993; Helin et al., 1993; Krek et al., 1993), this region beingconserved in all DP and E2F family members isolated to date (Girling etal., 1994), thus allowing diverse combinatorial interactions to occur.

During cell cycle progression the association of pRb, p107 and p130occurs in a temporally-regulated manner, each protein having its owncharacteristic profile of interactions with DRTF1/E2F (Shirodkar et al.,1992; Schwarz et al., 1993; Cobrinik et al., 1993). From the E2F familymembers isolated to date, E2F-1, -2, and -3 recognise pRb (Ivey-Hoyle etal., 1993; Lees et al., 1993) and E2F-4 the p107 protein (Beijersbergenet al., 1994; Ginsberg et al., 1994), a likely explanation being thatthe temporal interactions of pocket proteins reflect the regulatedcomposition and/or availability of the E2F family member in the E2F/DPheterodimer.

Although in many types of cells DP-1 is a frequent DNA binding componentof DRTF1/E2F, being present in the varying forms which occur during cellcycle progression (Bandara et al., 1994), other DP family members, suchas DP-2, are expressed in a tissue-restricted fashion (Girling et al.,1994). It would appear likely therefore that the molecular compositionof DRTF1/E2F is influenced by cell cycle progression and the phenotypeof the cell.

The complexity of the E2F family of proteins has yet to be elucidated.In order to address this question we have performed a yeast-based twohybrid screen to define new members of the family. Here, we report theisolation and characterization of murine E2F-5, a new member of the E2Ffamily. E2F-5 interacts with all the known members of the DP family ofproteins. In mammalian cell extracts E2F-5 exists as a physiologicalheterodimer with DP-1, an interaction which results in co-operative DNAbinding and transcriptional activation through the E2F site. E2F-5possesses a potent trans-activation domain which is specificallyinactivated upon pocket-protein binding. The protein sequence andmolecular organisation of E2F-5 is more closely related to E2F-4 thanother members of the family, defining for the first time a subfamily ofE2F proteins which are functionally and structurally related.

Results

Isolation of E2F-5

In order to explore the diversity of the E2F family of proteins weemployed a yeast two hybrid-based strategy (Fields and Song, 1989) toidentify new members (FIG. 8). We chose to use DP-1 as the bait, sinceDP-1 is a physiological and frequent partner for E2F-family members(Bandara et al., 1993; Bandara et al., 1994). An activation domaintagged cDNA library prepared from a 14.5 d.p.c. mouse embryo (Chevrayand Nathans, 1992) was screened for hybrid proteins capable ofinteracting with LexA-DP-1. One of the clones identified encoded ahybrid protein which by several criteria specifically interacted withLexA-DP-1. Partial analysis of the cDNA sequence indicated extensivesimilarity to E2F family members, and thus a cDNA clone encoding thecomplete protein sequence was further isolated from an F9 EC cDNAlibrary. Comparison of the protein sequence with other members of theE2F family indicated that the cDNA clone encoded a novel member.Following the designation adopted for previously isolated E2F proteinsas E2F-1, -2, -3 and -4, we refer to this clone as E2F-5.

The predicted size of murine E2F-5 is 335 residues (FIG. 9a). Thisprediction is based on the position of the first potential initiatingmethionine, together with the extensive homology existing to the humanE2F-5 sequence in which translation initiates at the same methionine(Hijmans et al., submitted). E2F-5 contains extensive sequencesimilarity with the domains conserved between other E2F family members(Ivey-Hoyle et al., 1993; Lees et al., 1993; Beijersbegen et al., 1994;Ginsberg et al., 1994). For example, the DNA binding domain shows 48%identity with E2F-1 and 87% with E2F-4 (FIG. 9b and c). Within thisarea, a C-terminal sub-domain contains the only region of similaritywith members of the DP family (indicated in FIGS. 9b and c). Thisregion, which is known as the DEF box (Girling et al., 1994; Lam and LaThangue, 1994), is intimately involved in the formation of the DP/E2Fheterodimer (Bandara et al., 1993; Bandara and La Thangue, inpreparation). The residues conserved within the DEF box between DP andE2F proteins are also perfectly conserved within E2F-5 (FIG. 9c),underscoring the potential importance of this sub-domain in formation ofthe DP/E2F heterodimer.

Several additional regions are conserved between E2F-5 and the otherfamily members. The marked box (Lees et al., 1993) and pocket-proteinbinding domain show 58% and 50% identity to these regions in E2F-1 and75% and 72% to the same regions in E2F-4 (FIG. 9b and 9 c). Thepositions of the hydrophobic residues in the leucine zip region are alsoconserved with other E2F family members (FIG. 9c). Indeed. E2F-5 mayform a longer zip than E2F-1, -2 and -3 because hydrophobic residues inE2F-5 are in register with the heptad repeat at two further C-terminalpositions (L144 and V151; see FIG. 9c).

The features of E2F-5 suggest a closer relationship with E2F-4 ratherthan with E2F-1, -2 and -3. Its organisation resembles that of E2F-4 inthat the protein does not extend much further then the N-terminus of theDNA binding domain, and both proteins lack the N-terminal cyclin Abinding domain which occurs in the other E2Fs (FIG. 9b). Furthermore,the protein sequence comparison indicates that E2F-5 and -4 are morerelated to each other than either is to the remaining members of thefamily. This is particularly evident across the conserved domains, wheremany residues are common between E2F-5 and -4 but not between E2F-1, -2and -3 (FIG. 9c). Overall, E2F-5 contains 70% amino acid residuesidentical with E2F-4 and 38% with E2F-1. Thus, based on this similarity,E2F-5 and -4 represent one subfamily of the E2F family of proteinswhilst E2F-1, -2 and -3, because of their close similarity, representanother.

Binding and Transcriptional Co-operation with DP Family Members

Generic DRTF1/E2F DNA binding activity arises when a DP family memberinteracts with an E2F family member (La Thangue, 1994). For DP-1 andE2F-1, the interaction results in co-operative transcriptionalactivation, DNA binding and interaction with pRb (Bandara et al., 1993Helin et al., 1993; Krek et al., 1993). We were therefore interested todetermine whether E2F-5 could co-operate with DP family members.

To answer these questions, we first used the yeast two hybrid assay withdifferent DP molecules represented in the hybrid bait as LexA fusionproteins (FIG. 8). Either E2F-5 or E2F-1 were expressed as activationdomain (GAD) tagged hybrid proteins and the degree of transcriptionalactivation of a LexA reporter construct assessed by measuringβ-galactosidase activity. Both E2F-5 and E2F-1 were equally capable ofinteracting with all known members of the DP family of proteins, that isDP-1, -2, -3 and Drosophila DP (data not shown).

We next assessed if E2F-5 could co-operate with DP-1 to activatetranscription through the E2F binding site. For this we used a yeastassay in which E2F-5 and DP-1 were expressed together or alone and thetranscriptional activity of an E2F site reporter construct, p4xWT CYC1,measured (FIG. 10a). In previous studies, this assay has been used tomeasure the co-operation between E2F-1 and DP-1 (Bandara et al., 1993).The transcriptional activity of the reporter was not significantlyaffected following the expression of the DP-1 hybrid proteins and onlymarginally by the E2F-5 hybrid (FIG. 10b). However, when both wereexpressed together, reporter activity was stimulated greater than 6-fold(FIG. 10b). The activity of p4xMT CYC1, a derivative of p4xWT CYC1 whichcarries mutant E2F binding sites (FIG. 10a; Bandara et al., 1993), wasunaffected in the same conditions (data not shown). We concludetherefore that E2F-5 co-operates with DP-1.

Transcriptional Activation and Pocket Protein Regulation of E2F-5

The ability of E2F-5 to activate transcription was assessed in bothyeast and mammalian cells. To assay transcriptional activity in yeast, aC-terminal region (from residue 198 to 335) of E2F-5 was fused to LexA,and the activity of a reporter construct driven by LexA binding sitesassessed (FIG. 11a). The E2F-5 protein contains a potent transactivation domain since the activity of the reporter in the presence ofpLEX.E2F-5 was much greater than when the vector alone was expressed(FIG. 11b); similarly, LexA E2F-1 was capable of activatingtranscription (FIG. 11b). Thus, E2F-5 activates transcriptionefficiently in yeast.

To confirm these results in mammalian cells and assess the functionalconsequences of the interaction of pocket-proteins with E2F-5, we fusedthe same region of E2F-5 to the Gal4 DNA binding domain and usedtransient transfection assays to study the transcriptional activity of areporter construct driven by Gal4 binding sites, pGAL-CAT (FIG. 12a). In3T3 cells, E2F-5 activated transcription efficiently relative to theactivity of the Gal4 DNA binding domain alone (FIG. 12b) since thetranscriptional activity of pGAL-CAT was 15-fold greater in the presenceof pGAL-E2F-5 relative to pG4. Similar results were obtained in avariety of other cell types (data not shown), indicating that E2F-5contains a trans-activation domain which functions in mammalian cells.

We then used the transcriptional activity of E2F-5 to assess thefunctional consequences of an interaction with either pRb or p107. Ascontrols for wild-type pRb and p107, we studied the activity of RbA22, aprotein encoded by a naturally-occurring mutant allele of Rb which failsto interact with DRTF1/E2F (Zamanian and La Thangue, 1992) and theactivity of anti-sense p107 RNA (Zamanian and La Thangue, 1993). Neitherwild-type pRb or RbΔ22 significantly affected the activity of E2F-5,since in the presence of either pCMVHRb or pCMVHRbΔ22 the activity ofpGAL-CAT was not affected (FIG. 12b). In contrast, co-expressing p107(from pCMV107) with E2F-5 reduced the transcriptional activity of E2F-5,an effect which was specific since anti-sense p107 (from pCMV107AS) hadno effect (FIG. 12b). We conclude that p107 inactivates thetranscriptional activity of E2F-5 in mammalian cells. Using a similarexperimental strategy, the p130 protein was shown to inactivate thetranscriptional activity of E2F-5 (data not shown).

E2F-5 is a Physiological DNA Binding Component of DRTF1/E2F

In order to determine if E2F-5 is a physiological DNA binding componentof the generic DRTF1/E2F DNA binding activity defined in extractsprepared from mammalian cells, two different anti-E2F-5 peptide serawere prepared against distinct peptide sequences; both antiseraspecifically reacted with a GST-E2F-5 fusion protein (data not shown).

The effect of these antisera on DRTF1/E2F DNA binding activity wasstudied by gel retardation assays performed with extracts from murine F9embryonal carcinoma (F9 EC) cells. Previous studies have shown that DP-1is a frequent, possibly universal, component of the DRTF1/E2F DNAbinding activity in F9 EC cell extracts (Girling et al., 1993; Bandaraet al., 1993), an example of which is shown in FIG. 13 (tracks 1 to 4).Both anti-E2F-5 sera disrupted DRTF1/E2F DNA binding activity, an effectwhich was specific since it was not apparent in the presence of thehomologous peptide (FIG. 13, tracks 5 through to 12). Althoughanti-E2F-5 caused a significant decrease in DNA binding activity, theeffect was clearly less marked than that caused by anti-DP-1 (FIG. 13,compare tracks 1 through 4 to 5 through 12). This may be because E2F-5is present in a sub-population of DP-1/E2F heterodimers in F9 EC cellextracts, a situation contrasting with that observed for DP-1.

DNA Binding Properties of E2F-5

To study the DNA binding properties of E2F-5 we expressed and purifiedE2F-5 as a GST fusion protein. Consistent with previous results (Bandaraet al., 1993), GST-DP-1 co-operated with GST-E2F-1 in binding to the E2Fsite, although E2F-1 possessed significant DNA binding activity whenassayed alone (FIG. 14, compare tracks 1 through 4). In contrast, E2F-5alone possessed barely detectable DNA binding activity (FIG. 14, tracks5 through 7). However, the co-operation between E2F-5 and DP-1 wasconsiderably greater than between E2F-1 and DP-1 (FIG. 13, tracks 8through 10). Thus, E2F-5 and DP-1 co-operate in DNA binding activity.

Levels of E2F-5 RNA

We were interested to determine the levels of E2F-5 RNA in differentcell lines and, moreover, compare E2F-5 levels to other members of theE2F family. For this, RNA was prepared from asynchronous cultures of F9EC cells together with a variety of leukaemic cell lines, and the levelof E2F-5 RNA assessed by Northern blotting. The amount of E2F-5 RNAvaried considerably from cell line to cell line; F9 EC cells and some ofthe leukaemic cell lines, for example, DAUDI and RAGI expressed highlevels (FIG. 15, tracks 1, 7 and 8). In contrast, HL60 and TF1 containedlow levels of E2F-5 RNA (FIG. 15, tracks 4 and 6). This profile of E2F-5RNA levels differed considerably from the levels of E2F-1. For example,significant levels of E2F-1 RNA were present in K562, HL60 and TF-1cells in contrast to E2F-5 (FIG. 15, tracks 3,4 and 6). The conversesituation was apparent in EL4 cells where E2F-5 levels were high andE2F-1 low (FIG. 15, track 10). We conclude that E2F-5 RNA levels areinfluenced by the cell type, and that there is little correlationbetween the levels of E2F-5 and E2F-1 RNA.

Discussion

E2F-5 and E2F-4 are a Sub-family of E2F Proteins

We report the isolation and characterisation of the fifth member of theE2F family of proteins, E2F-5. Although many of the domains in E2F-5,such as the potential leucine zip, the marked box and the pocket proteinbinding region, are conserved with other members of the E2F family, thelack of N-terminal sequence outside of the DNA binding domain indicatesa structural organisation similar to E2F-4 (Beijersbergen et al., 1994;Ginsberg et al., 1994). The other members of the E2F family, E2F-1, -2,and -3, have extended N-termini within which a domain capable ofinteracting with cyclin A resides (Krek et al., 1994). It has beensuggested that the role of this domain is to recruit a cyclin A/cdk2kinase to the DP-1/E2F heterodimer which results in the subsequentphosphorylation of DP-1, the functional consequence being reduced DNAbinding activity of the DP-1/E2F heterodimer (Krek et al., 1994). Such amechanism may be important in regulating the transcriptional activity ofE2F site-dependent genes at later times during cell cycle progression.The absence of a cyclin A binding domain in E2F-5 (and E2F-4) suggeststhat the DNA binding activity of the E2F-5/DP-1 heterodimer may bedown-regulated through other mechanisms. Indeed, a possible scenariothrough which this could be achieved would be through the p107 and/orp130 proteins since the spacer region in these proteins can bind eithercyclin A/cdk2 or cyclin E/cdk2 complexes (Lees et al., 1993; Cobrinik etal., 1993; Hannon et al., 1993; Li et al., 1993). It is possible thatthese pocket proteins replace the role of cyclin A-binding E2F familymembers and recruit cyclin/cdk complexes to the DP/E2F heterodimer.

Comparison of the protein sequence of E2F-5 with other members of theE2F family indicated a closer relationship with E2F-4 than with E2F-1,-2, and -3. Interestingly, E2F-4 is the only known member of the familywhich is capable of interacting with p107 in physiological conditions(Beijersbergen et al., 1994; Ginsberg et al., 1994). We have shown thatthe transcriptional activity of E2F-5 can be inactivated by p107 or p130rather than by pRb. However, this may reflect the closer relationship ofp107 to p130 than with pRb (Ewen et al., 1991; Cobrinik et al., 1993; Liet al., 1993) and may not therefore entirely reflect physiologicalinteractions. Consistant with this idea is the result that human E2F-5has been shown to interact with p130 in physiological conditions(Hijmans et al., submitted).

Co-operation Between E2F-5 and DP-1

In DNA binding and transcriptional activity E2F-5 co-operated with DP-1.In these respects, E2F-5 possesses similar properties to other membersof the E2F family. However. it is interesting to note that theco-operation between E2F-5 and DP-1 was considerably greater than, forexample, the co-operation observed between E2F-1 and DP-1 in equivalentexperimental conditions (for example see FIG. 14). If this assayreflects functional properties within cells, then it is possible in anintracellular environment of excess DP-1 that equivalent increases inthe amount of E2F-5 and E2F-1 would result in a relatively greater levelof E2F-5/DP-1 DNA binding activity. Furthermore, if there are preferredtarget genes for particular E2F/DP heterodimers then these differencesin DNA binding activity may reflect differences in transcriptionalactivity.

The precise roles of the different E2F proteins in cell cycle controlhave yet to be established. However, it is possible that they regulatethe transcriptional activity of target genes during discrete phases ofcell cycle progression. For example, that E2F- 1, -2 and -3 interactwith pRb (Helin et al., 1992; Kaelin et al., 1992; Ivey-Hoyle et al.,1993; Lees et al., 1993) suggests they regulate cell cycle progressionthrough G1. In contrast, p107 associates with DRTF1/E2F towards the endof G1, peaking in S phase (Shirodkar et al., 1992) whilst p130associates preferentially during early cell cycle progression, mostlyduring G0 (Cobrinik et al., 1993). However, the levels of E2F-1 andE2F-5 in a variety of cell types suggest that the expression of E2Ffamily members is not only influenced by cell cycle phase but also byphenotype. It is possible that cells utilise a preferred subset of E2Fproteins which are most suited to their cell cycle requirements.

The molecular and functional characterisation of E2F-5 reported here,together with its interaction with DP family members, indicates that avariety of heterodimers between E2F and DP family members aretheoretically possible. A major goal of future studies will be tounderstand the physiological role of each of these transcription factorsin cell cycle control

Materials and Methods

Yeast strains, media and methods. Saccharomyces cerevisiae strains usedin this study were as follows: W3031a (Mata ade2-100 trpl-I leu2-3,112his3-11 ura3); CTY10-5d (Matα ade2 trpl-901 leu2-3,112 his3-200 gal4gal80 URA3::lexAop-lacZ) and PCY2 (Matα gal14 gal80 URA3::GAL1-lacZlys2-801 his3-200 trp1-63 leu2 ade2-101) and JZ1 (Jooss et al., 1994;Matα lys2-801 ade2-10 leu2Δ1 trpΔ63 his3Δ200 URA3:: lexAop-CYC1-lacZ.Yeast strains were propagated in YPD or YNB media and transformed usinga modification of the lithium acetate method. The colony colourβ-galactosidase activity assay was performed by conventional procedures.β-galactosidase activity of individual transformants was quantitated inmid-log phase cultures for at least three independent transformants.

Library DNA, plasmids and oligonucleotides. pPC67 is a 14.5 d.p.c. CD-1mouse embryo oligo dT-primed cDNA library fused downstream of yeastsequences encoding the trans activation domain of the GAL4 protein (GAD;Chevray and Nathans, 1992). Complete cDNA clones were isolated from aλZapII F9 EC library of directionally cloned poly dT-primed cDNA(Schöler et al., 1990).

Plasmid pTR27 is a derivative of pBTM116 (Bandara et al., 1993) in whichthe polylinker sequences have been extended; pLEX.DP-1, pGAD.E2F-1,p4xWT CYC1 and p4xMT CYC1 have been described previously (Bandara etal., 1993). pLEX(HIS).DP-1 encodes a fusion of the complete bacterialLexA protein with DP-1 (from amino acid residue 59 to 410) in theplasmid pLEX(HIS), a derivative of pBTM1 16 in which TRP1 has beenreplaced with HIS3. pGAD.E2F-5 contains the entire coding sequence ofE2F-5 expressed as a hybrid protein with the activation domain of theyeast GAL4 protein (768-881) in the plasmid pACTII (Durfee et al.,1993). pLEX.E2F-5 contains the E2F-5 coding sequence from amino acidresidue 198 to 335 expressed as a hybrid protein downstream of thecomplete coding sequence of the LexA protein in the plasmid pTR27.pLEX.E2F-1 carries full-length E2F-1 (1-437) in pTR27. Plasmid pG4(previously called pG4m polyII; Webster et al., 1989) encodes the GAL4DNA binding domain (1-148) under the control of the SV40 early promoter.Plasmid pGAL4.E2F-5 contains E2F-5 coding sequence from residue 198 to335 fused downstream of the GAL4 sequences in pG4. Plasmids pCMVHRb,pCMVHRbΔ22, pCMV107 and pCMV107AS have been described previously(Zamanian and La Thangue, 1992; 1993). Library screening. 40 μg pPC67library DNA was co-transformed into CTY10-5d with 40 μg pLEX(HIS).DP-1.Approximately 400,000 transformants growing on selective agar plateswere screened by the in situ filter paper β-galactosidase assay. Torescue the library plasmids, blue colonies were isolated and cured ofpLEX(HIS).DP-1 by growing to saturation in selective liquid media in thepresence of histidine. After replica-plating on selective minimal agar,plasmid DNA from Trp⁺His⁻ colonies that failed to give a blue colourwhen assayed for β-galactosidase was electroporated into E.coli HW87.Plasmids were recovered and retransformed into CTY10-5d with eitherpLEX(HIS).DP-1 or the control plasmid (pLEX(HIS)). A plasmid conferringa Trp⁺ phenotype that gave a blue colony colour only in the presence ofpLEX(HIS).DP-1 was selected for further analysis. To obtain afull-length cDNA, the insert was excised, radiolabelled and used toscreen approximately 10⁶ plaques from the λZapII F9EC library from whicha full length E2F-5 cDNA was isolated and rescued into pBluescript.

Transient transfection of 3T3 cells. Transfections and assays wereperformed by the conventional calcium phosphate precipitation method asdescribed previously (Zamanian and La Thangue, 1992). β-galactosidaseactivity derived from pCMV-βgal as an internal control was measured aspreviously described (Zamanian and La Thangue, 1992).

Antisera and gel retardation analysis. Rabbit antisera raised againsttwo distinct peptide sequences derived from E2F-5, referred to asanti-E2F-5(1), anti-E2F-5 (2) were prepared and assessed for effect onDRTF1/E2F DNA binding activity in F9 EC cell extracts as previouslydescribed (Girling et al., 1993). The E2F binding site was taken fromthe adenovirus E2a promoter (La Thangue et al., 1990). Either thehomologous (+) or an unrelated (−) peptide was added to the DNA bindingassay to assess specificity as described previously (Girling et al.,1993). The anti-DP-1 (24) antiserum was raised against a peptide derivedfrom the C-terminal sequence of DP-1. DNA binding assays performed withGST fusion proteins were as described previously (Bandara et al., 1993;1994). GST-DP-1, -E2F-1 (Bandara et al., 1993) and -E2F-5 (amino acidresidue 2 to 335) were expressed and purified according to conventionalprocedures.

Northern analysis. Northern analysis of RNA levels was performed on RNAprepared from the indicated cell lines by conventional procedures. TheE2F-5 probe contained 840 nucleotides extending into the 3′ untranslatedregion. The E2F-1 probe contained the entire coding sequence of the genegenerated by PCR and a probe for GAPDH served as an internal control.

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25 1748 base pairs nucleic acid double linear DNA (genomic) CDS 31..10681 GGGGCCCGAC CACCGCGGGG CCGGGACGCG ATG GCG GCG GCA GAG CCC GCG AGC 54Met Ala Ala Ala Glu Pro Ala Ser 1 5 TCG GGC CAG CAG GCG CCG GCA GGG CAGGGG CAG GGC CAG CGG CCG CCG 102 Ser Gly Gln Gln Ala Pro Ala Gly Gln GlyGln Gly Gln Arg Pro Pro 10 15 20 CCG CAG CCT CCG CAG GCG CAA GCC CCG CAGCCG CCC CCG CCG CCG CAG 150 Pro Gln Pro Pro Gln Ala Gln Ala Pro Gln ProPro Pro Pro Pro Gln 25 30 35 40 CTC GGG GGC GCG GGG GGC GGC AGC AGC AGGCAC GAG AAG AGC CTG GGG 198 Leu Gly Gly Ala Gly Gly Gly Ser Ser Arg HisGlu Lys Ser Leu Gly 45 50 55 CTG CTC ACT ACC AAG TTC GTG TCG CTG CTG CAGGAG GCC AAG GAC GGC 246 Leu Leu Thr Thr Lys Phe Val Ser Leu Leu Gln GluAla Lys Asp Gly 60 65 70 GTT CTG GAT CTC AAA GCG GCT GCT GAT ACT TTG GCTGTG AGG CAA AAA 294 Val Leu Asp Leu Lys Ala Ala Ala Asp Thr Leu Ala ValArg Gln Lys 75 80 85 AGG AGA ATT TAT GAT ATC ACC AAT GTC TTA GAG GGA ATTGAC TTG ATT 342 Arg Arg Ile Tyr Asp Ile Thr Asn Val Leu Glu Gly Ile AspLeu Ile 90 95 100 GAA AAA AAG TCA AAA AAC AGT ATC CAG TGG AAA GGT GTAGGT GCT GGC 390 Glu Lys Lys Ser Lys Asn Ser Ile Gln Trp Lys Gly Val GlyAla Gly 105 110 115 120 TGT AAT ACT AAA GAA GTC ATA GAT AGA TTA AGA TATCTT AAA GCT GAA 438 Cys Asn Thr Lys Glu Val Ile Asp Arg Leu Arg Tyr LeuLys Ala Glu 125 130 135 ATT GAA GAT CTA GAA CTG AAG GAA AGA GAA CTT GATCAG CAG AAG TTG 486 Ile Glu Asp Leu Glu Leu Lys Glu Arg Glu Leu Asp GlnGln Lys Leu 140 145 150 TGG CTA CAG CAA AGC ATC AAA AAT GTG ATG GAC GATTCC ATT AAT AAT 534 Trp Leu Gln Gln Ser Ile Lys Asn Val Met Asp Asp SerIle Asn Asn 155 160 165 AGA TTT TCC TAT GTA ACT CAT GAA GAC ATC TGT AATTGC TTT AAT GGT 582 Arg Phe Ser Tyr Val Thr His Glu Asp Ile Cys Asn CysPhe Asn Gly 170 175 180 GAT ACA CTT TTG GCC ATT CAG GCA CCT TCT GGT ACACAA CTG GAG GTA 630 Asp Thr Leu Leu Ala Ile Gln Ala Pro Ser Gly Thr GlnLeu Glu Val 185 190 195 200 CCC ATT CCA GAA ATG GGT CAG AAT GGA CAA AAGAAA TAC CAG ATC AAT 678 Pro Ile Pro Glu Met Gly Gln Asn Gly Gln Lys LysTyr Gln Ile Asn 205 210 215 CTA AAG AGT CAT TCA GGA CCT ATC CAT GTG CTGCTT ATA AAT AAA GAG 726 Leu Lys Ser His Ser Gly Pro Ile His Val Leu LeuIle Asn Lys Glu 220 225 230 TCG AGT TCA TCT AAG CCC GTG GTT TTT CCT GTTCCC CCA CCT GAT GAC 774 Ser Ser Ser Ser Lys Pro Val Val Phe Pro Val ProPro Pro Asp Asp 235 240 245 CTC ACA CAG CCT TCC TCC CAG TCC TTG ACT CCAGTG ACT CCA CAG AAA 822 Leu Thr Gln Pro Ser Ser Gln Ser Leu Thr Pro ValThr Pro Gln Lys 250 255 260 TCC AGC ATG GCA ACT CAA AAT CTG CCT GAG CAACAT GTC TCT GAA AGA 870 Ser Ser Met Ala Thr Gln Asn Leu Pro Glu Gln HisVal Ser Glu Arg 265 270 275 280 AGC CAG GCT CTG CAG CAG ACA TCA GCT ACAGAT ATA TCT TCA GCA GGA 918 Ser Gln Ala Leu Gln Gln Thr Ser Ala Thr AspIle Ser Ser Ala Gly 285 290 295 TCT ATT AGT GGA GAT ATC ATT GAT GAG TTAATG TCT TCT GAC GTG TTT 966 Ser Ile Ser Gly Asp Ile Ile Asp Glu Leu MetSer Ser Asp Val Phe 300 305 310 CCT CTC TTA AGG CTT TCT CCT ACC CCG GCAGAT GAC TAC AAC TTT AAT 1014 Pro Leu Leu Arg Leu Ser Pro Thr Pro Ala AspAsp Tyr Asn Phe Asn 315 320 325 TTA GAT GAT AAC GAA GGA GTT TGT GAT CTGTTT GAT GTC CAG ATA CTA 1062 Leu Asp Asp Asn Glu Gly Val Cys Asp Leu PheAsp Val Gln Ile Leu 330 335 340 AAT TAT TAGATTCCAT GGAAACTTGG GACTGTTATCTACCTCTAAC TGTGTAACAT 1118 Asn Tyr 345 TTTAGACTTC TTAATAACCT AAATATTTAAAATAATGAAT GTAACACCTT TTTTAGTTCA 1178 CTGATTCTGA AGTGTTCTTC CCTAATACTTTCTTTACTTC ACAAAACTTC AACCATAAAA 1238 ACAAAGGGCT CTGATTGCTT TAGGGGATAAGTGATTTAAT ATTCACAAAC GTCCCCACTC 1298 CCAAAAGTAA CTATATTCTG GATTTCAACTTTTCTTCTAA TTGTGAATCC TTCCGTTTTT 1358 TCTTCTTAAG GAGGAAAGTT AAAGGACACTACAGGTCATC AAAAACAAGT TGGCCAAGGA 1418 CTCATTACTT GTCTTATATT TTTACTGCCACTAAACTGCC TGTATTTCTG TATGTCCTTC 1478 TATCCAAACA GACGTTCACT GCCACTTGTAAAGTGAAGGA TGTAAACGAG GATATATAAC 1538 TGTTTCAGTG AACAGATTTT GTGAAGTGCCTTCTGTTTTA GCACTTTAAG TTTATCACAT 1598 TTTGTTGACT TCTGACATTC CACTTTCCTAGGTTATAGGA AAGATCTGTT TATGTAGTTT 1658 GTTTTTAAAA TGTGCCAATG CCTGTACATTAACAAGATTT TTAAAAATAA AATTGTATAA 1718 AACATTAAAA AAAAAAAAAA AAAAAAAAAA1748 346 amino acids amino acid linear protein 2 Met Ala Ala Ala Glu ProAla Ser Ser Gly Gln Gln Ala Pro Ala Gly 1 5 10 15 Gln Gly Gln Gly GlnArg Pro Pro Pro Gln Pro Pro Gln Ala Gln Ala 20 25 30 Pro Gln Pro Pro ProPro Pro Gln Leu Gly Gly Ala Gly Gly Gly Ser 35 40 45 Ser Arg His Glu LysSer Leu Gly Leu Leu Thr Thr Lys Phe Val Ser 50 55 60 Leu Leu Gln Glu AlaLys Asp Gly Val Leu Asp Leu Lys Ala Ala Ala 65 70 75 80 Asp Thr Leu AlaVal Arg Gln Lys Arg Arg Ile Tyr Asp Ile Thr Asn 85 90 95 Val Leu Glu GlyIle Asp Leu Ile Glu Lys Lys Ser Lys Asn Ser Ile 100 105 110 Gln Trp LysGly Val Gly Ala Gly Cys Asn Thr Lys Glu Val Ile Asp 115 120 125 Arg LeuArg Tyr Leu Lys Ala Glu Ile Glu Asp Leu Glu Leu Lys Glu 130 135 140 ArgGlu Leu Asp Gln Gln Lys Leu Trp Leu Gln Gln Ser Ile Lys Asn 145 150 155160 Val Met Asp Asp Ser Ile Asn Asn Arg Phe Ser Tyr Val Thr His Glu 165170 175 Asp Ile Cys Asn Cys Phe Asn Gly Asp Thr Leu Leu Ala Ile Gln Ala180 185 190 Pro Ser Gly Thr Gln Leu Glu Val Pro Ile Pro Glu Met Gly GlnAsn 195 200 205 Gly Gln Lys Lys Tyr Gln Ile Asn Leu Lys Ser His Ser GlyPro Ile 210 215 220 His Val Leu Leu Ile Asn Lys Glu Ser Ser Ser Ser LysPro Val Val 225 230 235 240 Phe Pro Val Pro Pro Pro Asp Asp Leu Thr GlnPro Ser Ser Gln Ser 245 250 255 Leu Thr Pro Val Thr Pro Gln Lys Ser SerMet Ala Thr Gln Asn Leu 260 265 270 Pro Glu Gln His Val Ser Glu Arg SerGln Ala Leu Gln Gln Thr Ser 275 280 285 Ala Thr Asp Ile Ser Ser Ala GlySer Ile Ser Gly Asp Ile Ile Asp 290 295 300 Glu Leu Met Ser Ser Asp ValPhe Pro Leu Leu Arg Leu Ser Pro Thr 305 310 315 320 Pro Ala Asp Asp TyrAsn Phe Asn Leu Asp Asp Asn Glu Gly Val Cys 325 330 335 Asp Leu Phe AspVal Gln Ile Leu Asn Tyr 340 345 1340 base pairs nucleic acid doublelinear DNA (genomic) CDS 16..1020 3 AGGGCCGCGG CGGTG ATG GCG GCG GCG GAGCCC ACG AGC TCT GCT CAG CCC 51 Met Ala Ala Ala Glu Pro Thr Ser Ser AlaGln Pro 350 355 ACG CCG CAG GCC CAG GCT CAG CCG CCG CCG CAT GGG GCG CCATCC TCG 99 Thr Pro Gln Ala Gln Ala Gln Pro Pro Pro His Gly Ala Pro SerSer 360 365 370 CAG CCG TCG CGG CGC TCG CGG GGG GGC AGC AGC CGG CAC GAGAAG AGC 147 Gln Pro Ser Arg Arg Ser Arg Gly Gly Ser Ser Arg His Glu LysSer 375 380 385 390 CTG GGC TTG CTT ACC ACC AAA TTC GTG TCG TTG CTG CAGGAG GCG CAG 195 Leu Gly Leu Leu Thr Thr Lys Phe Val Ser Leu Leu Gln GluAla Gln 395 400 405 GAC GGC GTC CTG GAT CTC AAA GCG GCT GCA GAT ACC TTGGCT GTG AGG 243 Asp Gly Val Leu Asp Leu Lys Ala Ala Ala Asp Thr Leu AlaVal Arg 410 415 420 CAA AAG CGA AGA ATT TAT GAT ATC ACC AAT GTC TTA GAGGGA ATT GAT 291 Gln Lys Arg Arg Ile Tyr Asp Ile Thr Asn Val Leu Glu GlyIle Asp 425 430 435 CTA ATT GAA AAA AAA TCA AAG AAC AGT ATC CAG TGG AAGGGT GTA GGT 339 Leu Ile Glu Lys Lys Ser Lys Asn Ser Ile Gln Trp Lys GlyVal Gly 440 445 450 GCT GGC TGT AAT ACT AAA GAA GTT ATC GAT AGA TTA AGGTGT CTT AAA 387 Ala Gly Cys Asn Thr Lys Glu Val Ile Asp Arg Leu Arg CysLeu Lys 455 460 465 470 GCT GAA ATT GAA GAT CTC GAA TTG AAG GAA AGA GAACTT GAC CAG CAG 435 Ala Glu Ile Glu Asp Leu Glu Leu Lys Glu Arg Glu LeuAsp Gln Gln 475 480 485 AAG TTG TGG CTA CAG CAA AGC ATC AAA AAT GTG ATGGAA GAC TCC ATT 483 Lys Leu Trp Leu Gln Gln Ser Ile Lys Asn Val Met GluAsp Ser Ile 490 495 500 AAT AAC AGA TTT TCT TAT GTA ACT CAC GAA GAC ATCTGC AAT TGC TTT 531 Asn Asn Arg Phe Ser Tyr Val Thr His Glu Asp Ile CysAsn Cys Phe 505 510 515 CAT GGT GAT ACA CTG TTG GCC ATT CAG GCA CCT TCTGGT ACA CAG CTG 579 His Gly Asp Thr Leu Leu Ala Ile Gln Ala Pro Ser GlyThr Gln Leu 520 525 530 GAA GTA CCT ATT CCA GAA ATG GGA CAG AAT GGA CAAAAG AAA TAC CAG 627 Glu Val Pro Ile Pro Glu Met Gly Gln Asn Gly Gln LysLys Tyr Gln 535 540 545 550 ATA AAT CTG AAG AGT CAC TCA GGG CCT ATC CATGTG CTA CTT ATA AAT 675 Ile Asn Leu Lys Ser His Ser Gly Pro Ile His ValLeu Leu Ile Asn 555 560 565 AAA GAG TCC AGT TCA TCT AAG CCA GTG GTT TTTCCT GTT CCC CCA CCT 723 Lys Glu Ser Ser Ser Ser Lys Pro Val Val Phe ProVal Pro Pro Pro 570 575 580 GAT GAC CTC ACA CAG CCT TCC TCC CAG TCC TCAACT TCA GTG ACT CCA 771 Asp Asp Leu Thr Gln Pro Ser Ser Gln Ser Ser ThrSer Val Thr Pro 585 590 595 CAG AAA TCC ACC ATG GCT GCT CAA AAC CTG CCTGAG CAG CAT GTT TCC 819 Gln Lys Ser Thr Met Ala Ala Gln Asn Leu Pro GluGln His Val Ser 600 605 610 GAA AGA AGC CAG ACT TTC CAG CAG ACA CCA GCTGCA GAA GTA TCT TCA 867 Glu Arg Ser Gln Thr Phe Gln Gln Thr Pro Ala AlaGlu Val Ser Ser 615 620 625 630 GGA TCT ATT AGT GGA GAC ATC ATT GAT GAACTG ATG TCT TCT GAT GTG 915 Gly Ser Ile Ser Gly Asp Ile Ile Asp Glu LeuMet Ser Ser Asp Val 635 640 645 TTT CCT CTT TTA CGG CTT TCT CCT ACC CCAGCA GAT GAC TAC AAC TTT 963 Phe Pro Leu Leu Arg Leu Ser Pro Thr Pro AlaAsp Asp Tyr Asn Phe 650 655 660 AAT TTA GAT GAT AAT GAA GGA GTT TGT GATCTG TTT GAT GTT CAG ATA 1011 Asn Leu Asp Asp Asn Glu Gly Val Cys Asp LeuPhe Asp Val Gln Ile 665 670 675 CTA AAT TAT TAGATTCCAT GGAAACTTGGGACTATTATC TACCTCTATA 1060 Leu Asn Tyr 680 ACATTTTAGA ATTCTTTAATAACCTAAGTA TTTAAAATTA TGAATGTAAC ACCTTTTTAG 1120 TTCACTGATT CTGAAGTGTTCTTCCCTAAC ATTTTATTTT TTACTTCACA AAACTTGAAA 1180 GGGATATGCT GCTTCTGGGGGGTAGAGGTA AGATTACCTG TCCAGCAGCT GCCCCTCCAG 1240 TGACCACATT CAGTTTCTTTCAGTAGCTTC CTCTCCTGAG AGGCAGTTAC AGCAGGCTCA 1300 GTTCATCCAA ACAAAACATTGTCAGAAGTA CACTTATTTG 1340 335 amino acids amino acid linear protein 4Met Ala Ala Ala Glu Pro Thr Ser Ser Ala Gln Pro Thr Pro Gln Ala 1 5 1015 Gln Ala Gln Pro Pro Pro His Gly Ala Pro Ser Ser Gln Pro Ser Arg 20 2530 Arg Ser Arg Gly Gly Ser Ser Arg His Glu Lys Ser Leu Gly Leu Leu 35 4045 Thr Thr Lys Phe Val Ser Leu Leu Gln Glu Ala Gln Asp Gly Val Leu 50 5560 Asp Leu Lys Ala Ala Ala Asp Thr Leu Ala Val Arg Gln Lys Arg Arg 65 7075 80 Ile Tyr Asp Ile Thr Asn Val Leu Glu Gly Ile Asp Leu Ile Glu Lys 8590 95 Lys Ser Lys Asn Ser Ile Gln Trp Lys Gly Val Gly Ala Gly Cys Asn100 105 110 Thr Lys Glu Val Ile Asp Arg Leu Arg Cys Leu Lys Ala Glu IleGlu 115 120 125 Asp Leu Glu Leu Lys Glu Arg Glu Leu Asp Gln Gln Lys LeuTrp Leu 130 135 140 Gln Gln Ser Ile Lys Asn Val Met Glu Asp Ser Ile AsnAsn Arg Phe 145 150 155 160 Ser Tyr Val Thr His Glu Asp Ile Cys Asn CysPhe His Gly Asp Thr 165 170 175 Leu Leu Ala Ile Gln Ala Pro Ser Gly ThrGln Leu Glu Val Pro Ile 180 185 190 Pro Glu Met Gly Gln Asn Gly Gln LysLys Tyr Gln Ile Asn Leu Lys 195 200 205 Ser His Ser Gly Pro Ile His ValLeu Leu Ile Asn Lys Glu Ser Ser 210 215 220 Ser Ser Lys Pro Val Val PhePro Val Pro Pro Pro Asp Asp Leu Thr 225 230 235 240 Gln Pro Ser Ser GlnSer Ser Thr Ser Val Thr Pro Gln Lys Ser Thr 245 250 255 Met Ala Ala GlnAsn Leu Pro Glu Gln His Val Ser Glu Arg Ser Gln 260 265 270 Thr Phe GlnGln Thr Pro Ala Ala Glu Val Ser Ser Gly Ser Ile Ser 275 280 285 Gly AspIle Ile Asp Glu Leu Met Ser Ser Asp Val Phe Pro Leu Leu 290 295 300 ArgLeu Ser Pro Thr Pro Ala Asp Asp Tyr Asn Phe Asn Leu Asp Asp 305 310 315320 Asn Glu Gly Val Cys Asp Leu Phe Asp Val Gln Ile Leu Asn Tyr 325 330335 74 amino acids amino acid linear peptide 5 Lys Ser Pro Gly Glu LysSer Arg Tyr Glu Thr Ser Leu Asn Leu Thr 1 5 10 15 Thr Lys Arg Phe LeuGlu Leu Leu Ser His Ser Ala Asp Gly Val Val 20 25 30 Asp Leu Asn Trp AlaAla Glu Val Leu Lys Val Gln Lys Arg Arg Ile 35 40 45 Tyr Asp Ile Thr AsnVal Leu Glu Gly Ile Gln Leu Ile Ala Lys Lys 50 55 60 Ser Lys Asn His IleGln Trp Leu Gly Ser 65 70 74 amino acids amino acid linear peptide 6 LysSer Pro Gly Glu Lys Thr Arg Tyr Asp Thr Ser Leu Asn Leu Leu 1 5 10 15Pro Lys Lys Phe Ile Tyr Leu Leu Ser Glu Ser Glu Asp Gly Val Leu 20 25 30Asp Leu Asn Trp Ala Ala Glu Val Leu Lys Val Gln Lys Arg Arg Ile 35 40 45Tyr Asp Ile Thr Asn Val Leu Glu Gly Ile Gln Leu Ile Arg Lys Lys 50 55 60Arg Lys Asn His Ile Gln Trp Val Gly Arg 65 70 74 amino acids amino acidlinear peptide 7 Lys Ser Pro Gly Glu Lys Thr Arg Tyr Asp Thr Ser Leu AsnLeu Leu 1 5 10 15 Thr Lys Lys Phe Ile Gln Leu Leu Ser Gln Ser Pro AspGly Val Leu 20 25 30 Asp Leu Asn Lys Ala Ala Glu Val Leu Lys Val Gln LysArg Arg Ile 35 40 45 Tyr Asp Ile Thr Asn Val Leu Glu Gly Ile His Leu IleLys Lys Lys 50 55 60 Ser Lys Asn His Val Gln Trp Met Gly Cys 65 70 69amino acids amino acid linear peptide 8 Ser Arg His Glu Lys Ser Leu AsnLeu Leu Thr Thr Lys Phe Val Gln 1 5 10 15 Leu Leu Gln Glu Ala Lys AspGly Val Leu Asp Leu Lys Leu Ala Ala 20 25 30 Asp Thr Leu Ala Val Arg GlnLys Arg Arg Ile Tyr Asp Ile Thr Asn 35 40 45 Val Leu Glu Gly Ile Gly LeuIle Glu Lys Lys Ser Lys Asn Ser Thr 50 55 60 Gln Trp Arg Gly Val 65 75amino acids amino acid linear peptide 9 Arg Ser Arg Gly Gly Ser Ser ArgHis Glu Lys Ser Leu Gly Leu Leu 1 5 10 15 Thr Thr Lys Phe Val Ser LeuLeu Gln Glu Ala Gln Asp Gly Val Leu 20 25 30 Asp Leu Lys Ala Ala Ala AspThr Leu Ala Val Arg Gln Lys Arg Arg 35 40 45 Ile Tyr Asp Ile Thr Asn ValLeu Glu Gly Ile Asp Leu Ile Glu Lys 50 55 60 Lys Ser Lys Asn Ser Ile GlnTrp Lys Gly Val 65 70 75 74 amino acids amino acid linear peptide 10 SerMet Lys Val Cys Glu Lys Gln Arg Lys Gly Thr Thr Ser Tyr Asn 1 5 10 15Glu Val Ala Asp Glu Leu Val Ala Glu Phe Ser Ala Ala Asp Asn His 20 25 30Ile Leu Pro Asn Glu Ser Ala Tyr Asp Gln Lys Asn Ile Arg Arg Arg 35 40 45Val Tyr Asp Ala Leu Asn Val Leu Met Ala Met Asn Ile Ile Ser Lys 50 55 60Glu Lys Lys Glu Ile Lys Trp Ile Gly Leu 65 70 29 amino acids amino acidlinear peptide 11 Leu Thr Gln Asp Leu Arg Gln Leu Gln Glu Ser Glu GlnGln Leu Asp 1 5 10 15 His Leu Met Asn Ile Cys Thr Thr Gln Leu Arg LeuLeu 20 25 29 amino acids amino acid linear peptide 12 Leu Gly Gln GluLeu Lys Glu Leu Met Asn Thr Glu Gln Ala Leu Asp 1 5 10 15 Gln Leu IleGln Ser Cys Ser Leu Ser Phe Lys His Leu 20 25 29 amino acids amino acidlinear peptide 13 Leu Ser Lys Glu Val Thr Glu Leu Ser Gln Glu Glu LysLys Leu Asp 1 5 10 15 Glu Leu Ile Gln Ser Cys Thr Leu Asp Leu Lys LeuLeu 20 25 29 amino acids amino acid linear peptide 14 Leu Lys Ala GluIle Glu Glu Leu Gln Gln Arg Glu Gln Glu Leu Asp 1 5 10 15 Gln His LysVal Trp Val Gln Gln Ser Ile Arg Asn Val 20 25 29 amino acids amino acidlinear peptide 15 Leu Lys Ala Glu Ile Glu Asp Leu Glu Leu Lys Glu ArgGlu Leu Asp 1 5 10 15 Gln Gln Lys Leu Trp Leu Gln Gln Ser Ile Lys AsnVal 20 25 21 amino acids amino acid linear peptide 16 Asn Phe Gln IleSer Leu Lys Ser Lys Gln Gly Pro Ile Asp Val Phe 1 5 10 15 Leu Cys ProGlu Glu 20 21 amino acids amino acid linear peptide 17 Asn Leu Gln IleTyr Leu Lys Ser Thr Gln Gly Pro Ile Glu Val Tyr 1 5 10 15 Leu Cys ProGlu Glu 20 21 amino acids amino acid linear peptide 18 Ser Leu Gln IleHis Leu Ala Ser Ile Gln Gly Pro Ile Glu Val Tyr 1 5 10 15 Leu Cys ProGlu Glu 20 21 amino acids amino acid linear peptide 19 Lys Tyr Gln IleHis Leu Lys Ser Val Ser Gly Pro Ile Glu Val Leu 1 5 10 15 Leu Val AsnLys Glu 20 21 amino acids amino acid linear peptide 20 Lys Tyr Gln IleAsn Leu Lys Ser His Ser Gly Pro Ile His Val Leu 1 5 10 15 Leu Ile AsnLys Glu 20 19 amino acids amino acid linear peptide 21 Ala Leu Asp TyrHis Phe Gly Leu Glu Glu Gly Glu Gly Ile Arg Asp 1 5 10 15 Leu Phe Asp 19amino acids amino acid linear peptide 22 Gln Asp Asp Tyr Leu Trp Gly LeuGlu Ala Gly Glu Gly Ile Ser Asp 1 5 10 15 Leu Phe Asp 19 amino acidsamino acid linear peptide 23 Gln Glu Asp Tyr Leu Leu Ser Leu Gly Glu GluGlu Gly Ile Ser Asp 1 5 10 15 Leu Phe Asp 19 amino acids amino acidlinear peptide 24 Asp His Asp Tyr Ile Tyr Asn Leu Asp Glu Ser Glu GlyVal Cys Asp 1 5 10 15 Leu Phe Asp 18 amino acids amino acid linearpeptide 25 Asp Asp Tyr Asn Phe Asn Leu Asp Asp Asn Glu Gly Val Cys AspLeu 1 5 10 15 Phe Asp

What is claimed is:
 1. An isolated E2F-5 polypeptide selected from thegroup of: the polypeptide comprising SEQ ID NO:2; the polypeptidecomprising SEQ ID NO:4; a polypeptide comprising a fragment of SEQ IDNO:2 of at least 60 amino acids, said fragment being capable of forminga transactivation complex with a DP protein; and a polypeptidecomprising a fragment of SEQ ID NO:4 of at least 60 amino acids, saidfragment being capable of forming a transactivation complex with a DPprotein.
 2. An isolated polypeptide comprising SEQ ID NO:2.
 3. Anisolated polypeptide comprising SEQ ID NO:4.
 4. An Isolated polypeptidecomprising a fragment of at least 60 amino acids of the isolatedpolypeptide of claim 2 or 3 said fragment being capable of forming atransactivation complex with a DP protein.
 5. The isolated polypeptideof claim 1 that is detectably labeled.
 6. The isolated polypeptide ofclaim 1 fixed to a solid phase.
 7. A composition comprising thepolypeptide according to claim 1 together with a carrier or diluent. 8.A screening assay for identifying an inhibitor of E2F-5/DP complexformation, which assay comprises: bringing into contact: (i) a DPpolypeptide, said DP polypeptide being a component of an E2Ftranscription factor; (ii) the E2F-5 polypeptide of claim 1; and (iii) aputative inhibitor; under conditions in which the components (i) and(ii) in the absence of said putative inhibitor are able to form acomplex; and determining the extent to which, if any, the presence ofsaid putative inhibitor is able to disrupt the formation of the complex.9. The screening assay of claim 8 wherein said determining is made byexamining the ability of said complex to bind or activate an E2F DNAbinding site in vitro.
 10. The screening assay of claim 8 wherein theputative inhibitor is a fragment of 10 or more amino acids of thepolypeptide of claim
 2. 11. An assay according to claim 8 which furthercomprises selecting as an inhibitor of E2F-5/DP complex formation acompound capable of so disrupting said complex formation.